Acta Agriculturae Zhejiangensis

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2022, 34(8): 0-0.

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Analysis and evaluation of phenotypic diversities of early indica rice germplasm resources in Zhejiang Province

YANG Hailong, WANG Hui, LEI Jinchao, CAI Jinyang

2022, 34(8): 1571-1581. DOI: 10.3969/j.issn.1004-1524.2022.08.01

Abstract ( 381 )

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In order to clarify the genetic diversity of early indica rice germplasm resources in Zhejiang Province and improve the utilization efficiency of parental selection in early indica rice breeding, the diversity levels of 14 main phenotypic characters of 100 early indica rice germplasm resources were analyzed by means of coefficient of variation, genetic diversity index, principal component analysis, two-dimensional ranking analysis, stepwise regression analysis and cluster analysis. The results showed that the coefficient of variation of 14 phenotypic traits ranged from 0.96% (growth period) to 41.07% (shriveld grains per panicle). The genetic diversity index of 14 phenotypic traits ranged from 5.50 (shriveld grains per panicle) to 5.61 (growth period). Based on the two-dimensional sorting analysis of principal components, the germplasm resources with large panicles, small number of shriveled grains per panicle, small length-width ratio, moderate length of the second leaf and moderate growth period, long panicles and more effective panicles per plant were screened. According to the two-dimensional sorting results, early indica rice materials 4128, 4209, 4225 and 4278 were overlapping germplasms in two-dimensional sorting, which could be used as breeding parents or intermediate materials. Principal component analysis and comprehensive evaluation showed that the comprehensive traits of material 4021 ranked first among 100 early indica rice germplasm resources. At the same time, six phenotypic traits, such as filled grain number per panicle, shriveled grain number per panicle, seed setting rate, effective panicle per plant, grain length and grain width, could be selected as key indicators for comprehensive evaluation of early indica rice resources. At the same time, the hierarchical clustering was conducted based on the sum of squares of deviations, and 100 early indica rice materials were divided into four categories, with significant differences in various phenotypic traits.

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Influences of cadmium stress on seedling growth of different sweet corn inbred lines and screening of associated molecular markers via simple sequence repeats

JIANG Haoliang, HUANG Yun, LIANG Shaofang, XIE Mengchen, XU Tiancheng, SONG Zhiting, XIANG Wenwen, CHEN Qingchun, WAN Xiaorong, SUN Wei

2022, 34(8): 1582-1590. DOI: 10.3969/j.issn.1004-1524.2022.08.02

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In order to explore the responses of different sweet corn inbred lines to cadmium at seedling stage and to screen molecular markers associated with these responses, 13 sweet corn inbred lines were treated with 0.01 mol·L^-1 cadmium, comprehensive evaluation of these sweet corn inbred lines was carried out based on physiological indexes, and the associated molecular makers were identified via genomic simple sequence repeats (SSR) markers. It was shown that KY188 and M114A were sensitive materials, and T96-4, M3, T35 and T8-1 were tolerant materials by Cd tolerance analysis. Compared with the control (without Cd), the chlorophyll content of KY188 (Cd sensitive inbred line) treated with Cd significantly (P<0.05) decreased with time, and its plant height, root number, root length, fresh weight of shoot, dry weight of shoot, fresh weight of root, dry weight of root, total fresh weight of plant, total dry weight of plant were significantly (P<0.05) decreased by 18.87%, 24.32%, 21.13%, 58.86%, 2.04%, 33.77%, 26.11%, 47.11% and 14.29%, respectively. However, the tolerant inbred line T96-4 showed no significant changes in the above indexes after Cd treatment. Through correlation analysis, 9 SSR markers were screened out on the 2nd, 3rd, 4th, 5th, 6th, 8th, 9th and 10th chromosome.

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Identification and bioinformatics analysis of sucrose transporter family FtSUCs in Tartary buckwheat

LIANG Chenggang, WANG Yan, GUAN Zhixiu, WEI Chunyu, DENG Jiao, HUANG Juan, MENG Ziye, SHI Taoxiong

2022, 34(8): 1591-1598. DOI: 10.3969/j.issn.1004-1524.2022.08.03

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Plant sucrose transporter family proteins (SUC) are crucial carriers that mediate sucrose transmembrane transport. In this work, a total of 10 FtSUC genes were screened and identified based on the database of transcriptome sequencing in developing seedling of Tartary buckwheat, of which gene and encoding protein sequence were 1 436-7 109 bp and 148-605 aa, respectively. Eight FtSUCs were predicted to be located in the endoplasmic reticulum, of which sequence consistency was 34.30% including 20 conserved locis. Tartary buckwheat FtSUCs and Arabidopsis AtSUCs were classified into three types, including four FtSUCs in types Ⅰ, two FtSUCs in types Ⅱ and four FtSUCs in types Ⅲ. Tartary buckwheat FTSUCs proteins were mainly composed of α-helix, irregular coil, elongated chain and β-fold. Among them, 9 FtSUCs contain transmembrane domains. The expressing patterns of FtSUCs varied in developing stem of Tartary buckwheat seedlings. For these genes, FtPinG0002608200.01, FtPinG0001943900.01 and FtPinG0001944100.01 were highly expressed at cotyledon stage, while FtPinG0000342600.01 showed gradually increased gene expressing pattern, whereas FtPinG0001943500.01 and FtPinG0009584000.01 exhibited stable gene expressing pattern during seedling development. It was speculated that the varied gene expressing pattern of FtSUCs might perform specific functions at different developmental stages. Correlation analysis confirmed that there were significantly positive or negative correlations among several FtSUCs genes in Tartary buckwheat, suggesting that the synergistic or complementary effects might be occurred among FtSUCs genes, which together regulated the sucrose transmembrane.

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Isolation, identification and biological characteristics of rabbit-derived Escherichia coli bacteriophage

WANG Zhipeng, ZHAO Jian, HUANG Pan, CUI Xuemei, NAN Li, SONG Houhui, BAO Guolian, LIU Yan

2022, 34(8): 1599-1608. DOI: 10.3969/j.issn.1004-1524.2022.08.04

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In this study, one enteropathogenic Escherichia coli strain was isolated and identified via Gram staining and PCR from a rabbit farm in Zhejiang Province, China, which was named as ZJE1. ZJE1 was sensitive to streptomycin and ciprofloxacin. Four bacteriophages were isolated with the ZJE1 strain, which were named as ZRP2, ZRP3, ZRP4 and ZRP5, respectively. The biological and morphological characteristics of these bacteriophages were analyzed. It was shown that the bacteriophages were composed of hexagonal head, contractible tail sheath and tail tube. All the four bacteriophages belonged to Myoviridae family. The host spectrum, optimal multiplicity of infection (MOI), pH stability, thermal stability and one-step growth curve of these four bacteriophages were determined by double-layer plate method. The results showed that ZRP2, ZRP3, ZRP4 and ZRP5 all showed good activity under 37-55 ℃ with high host specificity. ZRP2, ZRP3 could maintain good activity under pH value of 7, while ZRP4 and ZRP5 could maintain good activity under pH value of 5-7. ZRP2, ZRP3, ZRP5 showed good lytic activity in vitro, with incubation period of 11-19 min. These findings provided research basis for the treatment of rabbit enteropathogenic E. coli with bacteriophage.

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Verbascoside lowers Streptococcus suis serotype 2 pathogenicity in mice by inhibiting hemolytic activity of suilysin

ZHAN Jiafei, XU Kui, ZHANG Lei, XIA Jieying, HONG Yang, DONG Han, LIU Yanglu, ZHOU Jing, YUAN Mingming, WANG Yongjin, YAN Liangchun

2022, 34(8): 1609-1616. DOI: 10.3969/j.issn.1004-1524.2022.08.05

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The aim of this study is to explore the effect of verbascoside (VBS), a natural compound, on the pathogenicity of Streptococcus suis serotype 2 (SS2). By determining the minimum inhibitory concentration (MIC), growth curve of SS2 and hemolysis activity of erythrocytes, is was found that the low concentration of VBS significantly inhibited the hemolytic activity of SS2 without interfering with its growth. Western blotting showed that VBS had no influence on the expression of suilysin (SLY) in SS2. It was predicted by molecular docking that VBS could be embedded into the domain 4 of SLY, which was through the formation of three hydrogen bonds between VBS and two residues (SER-388, GLN-441) of SLY. And it was further confirmed that VBS directly inhibited the hemolytic activity of SLY in vitro. Moreover, VBS significantly increased the survival rate of mice infected with SS2 in vivo. In conclusion, without affecting the growth of SS2, VBS can inhibit the hemolytic activity of SLY to reduce the pathogenicity of SS2 in mice.

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Analysis on pathogens of frogs with crooked head, broken head or white eye

LI Xudong, LIU Yongtao, YANG Xianle, YANG Yibin, AI Xiaohui

2022, 34(8): 1617-1625. DOI: 10.3969/j.issn.1004-1524.2022.08.06

Abstract ( 407 )

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In order to investigate the causes of a series of frog diseases in the main breeding areas of Hubei and Fujian, more than 40 strains of dominant bacteria were isolated from the brain and internal organs of frogs with typical symptoms of crooked head, broken head or white eyes. Through 16S rRNA gene sequence alignment, it was found that 40 dominant strains were homologous with Elizabethkingia miricola. After physiological and biochemical identification and phylogenetic analysis, the genetic differences were found in 40 dominant strains, the cluster analysis of 40 strains showed that host and region were important factors affecting genetic relationship, and the typical strain QW08 was identified as E. miricola. By regression infection experiment, strain QW08 was confirmed to be the pathogen of crooked head, broken head and white eye disease in frogs. Histopathological observations showed that E. miricola infection could cause damage to multiple organs and tissues in the whole body, including brain, intestinal tract, liver, spleen and kidney. The main pathological manifestations were degeneration, necrosis and inflammation, leading to dysfunction of multiple organs and tissues and death of infected frogs. Drug sensitivity analysis showed that QW08 was only highly sensitive to florfenicol, and showed different degrees of resistance to the other 19 antibiotics. The results provided a theoretical basis for the prevention and control of the disease in frogs.

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Effect of reducing chemical fertilizer and applying bio-organic fertilizer on yield and quality of Chinese cabbage in open field

RU Chao, YU Jihua, WU Yue, FENG Zhi, GOU Zhaohui, JIN Ning, WANG Shuya, LIU Zeci, LYU Jian

2022, 34(8): 1626-1637. DOI: 10.3969/j.issn.1004-1524.2022.08.07

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In order to improve Chinese cabbage's problems of reduced yield, poor quality and low fertilizer utilization caused by excessive use of chemical fertilizers, Wangchun Chinese cabbage cultured in the open field was used as the test material, and no fertilization (CK1) and conventional fertilization (CK2), chemical fertilizer balance fertilization (T1), chemical fertilizer reduction of 30%+6 000 kg·hm^-2 biological organic fertilizer (T2), chemical fertilizer reduction of 30%+9 000 kg·hm^-2 biological organic fertilizer (T3), chemical fertilizer reduction of 40%+6 000 kg·hm^-2 bio-organic fertilizer (T4) and chemical fertilizer reduction of 40%+9 000 kg·hm^-2(T5) were set up to investigate the effects of different fertilization treatments on the growth, yield and quality of Chinese cabbage. The results showed that the application of bio-organic fertilizer increased the root vigor and chlorophyll content of each treatment to varying degrees. Compared with the local conventional fertilization, the root vigor and chlorophyll content increased by 15.5%-40.3%, and the chlorophyll content increased by 11.5%-37.5%, and increased with the application of bio-organic fertilizer. Compared with CK2, T1-T5 treatment increased the yield by 0.04%-20.91%. T3 treatment increased the yield by 20.91%, and its economic benefit increased by 9.04%. The application of bio-organic fertilizer increased the fertilizer contribution rate, and the fertilizer was reduced by 30% and combined with bio-organic application. The content of soluble sugar, soluble protein, and free amino acid in T3 treatment were 0.91 percent points, 81.00% and 9.46% higher than that of CK2, respectively. Nitrate content in each treatment with bio-organic fertilizer was reduced by 11.53%-21.52%, V_C, total flavonoids, glucosinolate content in T3 treatment was significantly higher than CK2, the total phenol content of each treatment was not significantly different. Compared with CK2, the content of mineral elements in each treatment increased to different degrees, and the increase was more obvious after applying bio-organic fertilizer. The membership function analysis was carried out on 15 indicators, and the comprehensive evaluation of T3 treatment with 30% reduction in chemical fertilizer and 9 000 kg·hm^-2 bio-organic fertilizer had the best effect, followed by T2>T5>T1>T4>CK2>CK1.

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Creation of allotetraploid vegetable germplasm by interspecific hybridization of Brassica rapa L. ssp chinensis var. utilis Tsen et Lee and Brassica oleracea var. Alboglabra

LYU Fengxian, HE Jiangming, LI Chongjuan, YANG Ding, HU Jingfeng, YANG Hongli, LAN Mei, XU Xuezhong, ZHANG Liqin

2022, 34(8): 1638-1647. DOI: 10.3969/j.issn.1004-1524.2022.08.08

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Hybridization between Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee and Brassica oleracea var. Alboglabra can enrich genetic background and innovate germplasm resources. However, due to the dysplasia of fertilization zygote, embryo rescue of the interspecific hybrid is necessary to obtain the offspring. The interspecific hybridization was performed using Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee as female parents and Brassica oleracea var. Alboglabra as male parents. Hybrid embryos were harvested for artificial culture after crosses. In this experiment, five kinds of mediums, six different sampling time, four kinds of rooting mediums and three kinds of chromosome-doubling manner were set. Suitable interspecific hybrid embryo rescue methods, proliferation and expansion methods, and chromosome doubling methods were selected to improve the hybrid acquisition rate. The results showed that embryogenesis rate of ERM₄ (MS+0.5 mg·L^-1 6-BA+0.5 mg·L^-1 NAA+1.0 mg·L^-1 KT+1 g·L^-1 AC) was 10.47%, used as the optimal embryo rescue medium. 12 d after pollination was the best sampling time since the embryogenesis rate was 26.32%. RM₄ (1/2MS+0.5 mg·L^-1 IBA) had the highest rooting rate and the shortest rooting time, which was the best rooting medium. The method that applied at growth points 5 times with colchicine, was the best chromosome-doubling manner, in which chromosome doubling rate reached to 62.26%. And then the F₁ generation hybrids plants were identified through morphological identification, chromosome analysis and ploidy identification. The results showed that the hybrids obtained in the experiment were true hybrids, and the fertile plants were allotetraploidy. The successful creation of allotetraploid vegetable germplasm laid a foundation for subsequent breeding research.

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Effects of balanced fertilization on growth, quality, mineral elements contents and yield of tomato cultivated in substrate in greenhouse

LI Wangxiong, ZHANG Yang, TANG Zhongqi, YU Jihua

2022, 34(8): 1648-1660. DOI: 10.3969/j.issn.1004-1524.2022.08.09

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In order to clarify the effects of balanced fertilization on tomato cultivated in substrate in the greenhouse, the tomato (Solanum lycopersicum L.) cultivar 184 was used as test material. Four treatments were designed according to the principle of balanced fertilization of N, P and K, namely no fertilization (CK0), conventional fertilization (CK, 23.7 kg N, 38.4 kg P₂O₅ and 72.9 kg K₂O per 667 m²), balanced fertilization 1 (T1, 38.38 kg N, 16.45 kg P₂O₅ and 53.18 kg K₂O per 667 m²), balanced fertilization 2 (T2, 10% less fertilizer than T1, 33.58 kg N, 514.4 kg P₂O, 46.5 kg K₂O per 667 m²), effects of balanced fertilization on tomato growth, quality, yield and mineral element content were studied. The results showed that balanced fertilization could promote tomato growth, improve quality, mineral element content and yield in different degrees, balanced fertilization 1 (T1) had the best effect. Compared with CK, plant height, leaf number, maximum net photosynthetic rate and root activity of tomato in T1 treatment increased by 9.97%, 5.19%, 25.13% and 95.79% respectively; soluble solid, soluble sugar, iron, magnesium, calcium and yield in T1 treatment increased by 19.82%, 16.09%, 15.24%, 11.30%, 69.06% and 15.25%, respectively. The results of correlation and principal component analysis showed that there was a certain correlation and dependence between the quality and yield of tomato indexes. According to the final comprehensive score model, it was found that the score of T1 treatment was the highest and CK0 was the lowest, indicating that balanced fertilization had a great impact on the growth, quality, mineral elements contents and yield of tomato cultivated in substrate. Among them, the fresh weight of single fruit and contents of V_C, mineral elements, phosphorus, potassium, iron, manganese and zinc were the main factors affecting the growth and quality of tomato. Therefore, the balanced fertilization 1 treatment (T1) could be a fertilizer application pattern for rational allocation of fertilizer resources by enhancing the photosynthetic capacity of tomato leaves, increasing yield and improving fruit quality.

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Flowering transition of Fragaria pentaphylla under low-temperature and short-day conditions

XIANG Xi, WANG Siyue, PU Junhong, TANG Wenlu, CHEN Qing

2022, 34(8): 1661-1668. DOI: 10.3969/j.issn.1004-1524.2022.08.10

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Photoperiod and temperature are the two main factors that affect the flowering induction in plants. In order to study the effect of the combination of short-day and low temperature treatments on the flowering induction of Fragaria pentaphylla, four important determinants CONSTANS (FpCO), FLOWERING LOCUS T (FpFT), LEAFY (FpLFY), and APETALA1 (FpAP1) were analyzed. Gene expressions in different developmental stages of leaves, stem apex meristems, fruits, and after induction at different time points under low temperature (15 ℃) and short day (8 h·d^-1) were analyzed. At the same time, the apex meristem at different time points was stripped, fixed and paraffin embedded for microscopic observation of fluorescence differentiation process. The results showed that: FpFT1, FpLFY3, FpCO and FpAP1 were the key flower-forming factors in wild strawberries. The FpCO gene reached the peak of expression after 28 d of treatment, and then the expression level dropped sharply. Since 42 d, the overall expression level of FpFT1 gene was relatively high, after which it showed a downward trend, but maintained a high expression level. The FpAP1 and FpLFY3 gene expression trends were similar, both peaking at 42 d, and decreasing after the peak. These results implied that Fragaria pentaphylla started the flower induction process after four weeks of treatment under the tested condition and finished the transition process after 42 d. These results were consistent with the morphological differentiation process observed by slices.

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Dry matter and nutrients accumulation characteristics of high-yield and conventional daylily varieties

WANG Feng, LIU Haitian, YU Qiaogang, YE Jing, HE Xinhua, ZHOU Yang, MA Junwei

2022, 34(8): 1669-1678. DOI: 10.3969/j.issn.1004-1524.2022.08.11

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The study on nutrients absorption and its relationship with high-yield could provide theoretical basis for the reasonable fertilization and development of special fertilizer for daylily (Hemerocallis fulva). In the present study, conventional variety Shixin and high-yield variety 207 were used as test materials, and the dry matter accumulation dynamics, and contents of N, P, K, S, Ca, Mg, Zn, B, Mo, Mn were determined at different growth stages during March to November in 2020. It was shown that the dry matter accumulation of both varieties reached the maximum at the budding and flowering stage, and the highest dry matter accumulation speed was from the leaf-expansion stage to the bolting stage. The dry weight of root of the conventional variety reached the highest at the bolting stage, while the high-yield variety reached the maximum at the budding and flowering stage. The requirement of N, P, K, S, Ca, Mg, Zn, B, Mo, Mn in the high-yield variety was significantly (P<0.05) higher than that in the conventional variety. The requirement of N, K, P of high-yield variety was mainly before the budding and flowering stage, while the absorption of N and K of the conventional variety was concentrated within the budding stage to the leaf-expanding stage, and the absorption of P was mainly in the bolting stage. In the budding stage, the absorption of S and Ca was the largest in the conventional variety, and the highest absorption of Mg was found in the leaf-expansion stage, while the absorption of S, Ca, Mg of the high-yield variety was mainly before the budding and flowering stage. The absorption of Zn, B, Mo and Mn of the conventional variety was concentrated before the bolting stage, yet the absorption of Zn, B, Mo and Mn of the high-yield variety lasted until the bolting stage, and the accumulation amount in the bolting stage was the highest. In the whole growth period, the N, P, K demand ratio was 1.0∶0.25∶2.3 in the high-yield variety and 1.0∶0.24∶1.7 in the conventional variety; the demand ratio of S, Ca, Mg was 1.0∶11.5∶1.8 in the high-yield variety and 1.0∶5.5∶1.0 in the conventional variety. In conclusion, the high-yield variety had longer root development time and higher dry matter accumulation. In the preparation and development of special fertilizers for high-yield cultivation of day-lily, the supply of K and medium and trace elements should be ensured to meet the the growth and development requirement of daylily.

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Cloning and bioinformatics analysis of a PEBP family gene from Cymbidium faberi

CHU Zhigang, TIAN Yunfang

2022, 34(8): 1679-1691. DOI: 10.3969/j.issn.1004-1524.2022.08.12

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In order to explore the characteristics and function of PEBP gene family in Cymbidium faberi, the gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR) combined with rapid-amplification of cDNA ends (RACE). R language, CodonW, CHIPS and CUSP software were used to analyze the codon preference, and the protein structure and function of the gene were predicted by using bioinformatics tools. The results showed that the cDNA sequence of PEBP gene contained 531 bp open reading frame (ORF) and encoded 176 amino acids. The gene was named as CfPEBP(Accession No. MT795710). The molecular formula of CfPEBP was C₈₈₂H₁₃₆₆N₂₅₄O₂₆₀S₅ with molecular weight of 19 848.39 u, pI of 6.42, and phosphorylation sites of 15. CfPEBP protein has no signal peptide domain and has PEBP functional domain. Phylogenetic tree analysis showed that CfPEBP protein had the closest evolutionary distance with FT gene of Cymbidium goeringii, and the homology was 100%. Codon preference analysis showed that the codon preference of CfPEBP was not obvious; based on the relative synonymous codon usage (RSCU) value, GGC, AGA, AGU, AAG, CCA, CUC (RSCU≥2) were the most preferred codons. The RSCU value analysis of PEBP gene in 18 species showed that there were more than 25 codons with RSCU value>1 in Cymbidium faberi, Oncidium hybrid(KJ909968), Oncidium hybrid(EU583502), Dendrobium hybrid(MF063061), Musa acuminata(KF853468) and Ipomoea nil(AB154823). PEBP family genes had more preference than other codons for GGC, AGG, AGA, AGC, AGU, UGC, GAG, AAG, UAC, GCC, CCA, CUC. The relative expression level of CfPEBP gene was the highest in the leaves of Cymbidium faberi at flowering stage, followed by flower bud at bud stage and inflorescence rachis at full-blossom stage. This study provided the basis for further study on the function of CfPEBP gene.

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Development of plasmid standard material for detection of genetically modified carnation line Moonlite

YANG Rumeng, YIN Lu, QIAN Changyuan, ZUO Cuihua, YU Haiyan, LI Xiang

2022, 34(8): 1692-1702. DOI: 10.3969/j.issn.1004-1524.2022.08.13

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By the end of 2020 year, nineteen genetically modified (GM) carnation lines had been commercialized in the world. In order to detect and supervise GM carnations effectively, it is necessary to develop standard materials. Until now, there are no standard materials for GM carnation detection that have been reported around the world. Therefore, this study developed a plasmid standard molecule pLite for GM carnation line Moonlite, which has been commercialized and planted for a long time, so that it could be suitable for the application of the standard for GM carnation detection, and could support the inspection and supervision of GM products imported and exported. To develop a plasmid DNA molecule for Moonlite, two fragments of carnation endogenous ANS gene with 1 279 bp in length and the 5'-end event-specific sequence of GM carnation Moonlite with 406 bp in length were inserted into plasmid pcDNA3.0. The distance between the two inserted sites was more than 1 kb. Then, the plasmid DNA was diluted and packaged. The homogeneity, minimum sampling amount, short-term and long-term stability and reference values of the plasmid were measured, meanwhile, the uncertainty were evaluated. The genome sequencing results showed that the sequence of pLite was consistent with the published sequence. Results of the homogeneity test showed that F values of ANS gene and Moonlite event-specific sequence were 1.61 and 1.14, respectively, which were both less than the F value under 95% confidence interval, indicating that the plasmid DNA molecule was homogeneous. The minimum sampling amount of pLite was 2 μL. Results of stability experiments showed that pLite stayed stable at 45 ℃ for 14 days. At room temperature of 26 ℃ and below, the plasmid DNA molecule could be stably stored for one month, and could be stably stored for 6 months at 4 ℃ and below, indicating that pLite was suitable for transportation. After repeated freeze-thaw 25 times, the copy number of pLite had no significant change, which could meet the requirements of daily detection. The calculation results indicated that the reference values of ANS gene and Moonlite specific sequence were (6.13±0.22)×10⁶ μL^-1 and (6.10±0.24)×10⁶ μL^-1 in pLite, respectively. The developed plasmid DNA molecule pLite had good homogeneity and stability, at the same time it was suitable for the detection of GM carnation event Moonlite.

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Cloning of PhaSEP3 gene in Phalaenopsis and its expression in floral organ mutants

XU Shenping, ZHANG Yan, LIANG Fang, JIANG Suhua, NIU Suyan, CUI Bo, YUAN Xiuyun

2022, 34(8): 1703-1712. DOI: 10.3969/j.issn.1004-1524.2022.08.14

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The class E MADS-box is one of the essential genes in molecular models of floral organ development. Studying its expression pattern in mutants will provide basis for understanding the molecular mechanism of flower organs in orchids and improving the regulation theory of flower development. In this study, a Class E MADS-box gene PhaSEP3 (GenBank accession number: MZ436812) was cloned from petal in Phalaenopsis using the method of rapid-amplification of cDNA ends (RACE), and the expression level of this gene in different tissues and five mutants of Phalaenopsis was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the cDNA full-length of PhaSEP3 gene was 1 236 bp with open reading frame (ORF) of 753 bp encoding 250 amino acids, and with SEPⅠ and SEPⅡ motifs in its C-terminal. The phylogenetic analysis indicated that the protein was highly homologous to PeSEP3 and AGL9 from Phalaenopsis. The expression analysis showed that PhaSEP3 gene was expressed mainly in the reproductive organs and ovary after pollination. In different floral organ mutants, the expression level of the PhaSEP3 gene was increased significantly in sepal and lip in the lip-like lateral sepal mutant. The expression level of this gene was decreased significantly in petal, lip and ovary, while was increased significantly in column in the mutant of degenerated stamen converting petals. The expression level of this gene was increased significantly in petal and lip in the lip-like lateral petals mutant, and was decreased significantly in petal along with increased significantly in column and ovary in the petal degeneration mutant. However, the expression level of PhaSEP3 gene was increased significantly in the sepal and petal in the mutant of lateral petals with anther. It was concluded that PhaSEP3 gene mainly regulated the development of floral tissues and ovary after pollination in Phalaenopsis. In the floral organ mutants, the class E gene might interact with other floral development genes to regulate the morphological development of floral organ. The results provided further understandings of the molecular mechanism of floral organ diversity in orchid.

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Resistance of 12 tea cultivars to Dendrothrips minowai Priesner and Empoasca Onukii Matsuda and a preliminary identification of resistant components

YANG Chun, MENG Zehong, LI Shuai, LIANG Sihui, QIAO Dahe, CHEN Zhengwu

2022, 34(8): 1713-1724. DOI: 10.3969/j.issn.1004-1524.2022.08.15

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This article aims to ascertain the resistance of 12 tea cultivars to Dendrothrips minowai Priesner and Empoasca Onukii Matsuda, and to clarify the related resistance biochemical components. Through a one-year field survey, the resistance performance of 12 tea cultivars to D. minowai and E. onukii were confirmed. HPLC was used to analyze caffeine, amino acids, catechin components and the resistance biochemical components were also defined through correlation analysis. Results showed, Wuniuzao, Jinguanyin, Zhongcha 108 were more susceptible to D. minowai. In contrast, Taixuan 0310 and Qianfu 4 possessed higher resistance. Fuding Dabaicha was more susceptible to E. onukii. In contrast, Qianmei 601 possessed higher resistance. The occurrence quantity of D. minowai was negatively significant correlated with caffeine content. Most amino acids contribute to the susceptibility of E. onukii. Arginine was positively significant correlated to the occurrence quantity of E. onukii. Except epigallocatechin, the other 5 catechin monomers contribute to the resistance of E. onukii. The content of (+)-catechin was negatively significant correlated to the occurrence quantity of E. onukii.

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Effects of different safe utilization technologies on cadmium reduction in rice-vegetable rotation system in northern Hainan, China

HUANG Feng, XING Jianping, FU Shaohuai, PAN Pan, WU Lin, LIU Beibei, CHEN Miao

2022, 34(8): 1725-1733. DOI: 10.3969/j.issn.1004-1524.2022.08.16

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In order to evaluate the effects of safe utilization technologies on Cd-contaminated farmland in Hainan, rice-vegetable rotation system in northern Hainan with light to moderate Cd pollution was selected as the study object. The effects of 6 safe utilization technologies on Cd reduction in pepper and rice, and the effects of 9 passivators on Cd accumulation in rice were compared. The results showed that the Cd reduction effects of various safe utilization technologies decreased in the order of optimal fertilization+soil conditioning+foliage resistance>soil conditioning+foliage resistance>optimal fertilization+foliage resistance>soil conditioning>foliage resistance>optimal fertilization, and the Cd reduction rates in pepper and rice were 61.6%-91.5%, 49.4%-91.3%, respectively. All these technologies reduced health risk of Cd-intake by pepper to an acceptable range, but only optimal fertilization+soil conditioning+foliage resistance and soil conditioning+foliage resistance reduced the health risk of Cd-intake by rice to an acceptable range. Applying passivators could significantly (P<0.05) reduce Cd content in rice, but the Cd reduction rates varied from 29.7% to 77.0%. Among them, biochar and phosphorus combined passivator (biochar+phosphate rock powder, biochar+activated phosphate rock powder), vermicompost and iron phosphorus combined passivator (vermicompost+zero-valent iron+phosphate rock powder) had better effect, and the Cd reduction rate of rice was higher than 68%. In conclusion, optimal fertilization+soil conditioning+foliage resistance, soil conditioning+foliage resistance were feasible for the safe utilization of Cd-contaminated farmland in Hainan, and biochar and phosphorus combined passivator, and vermicompost and iron phosphorus combined passivator were recommended passivators for soil conditioning.

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Effects of intestinal conditions and particle size on in vitro digestion of processed Carya cathayensis Sarg.

SHAO Yuchen, MU Honglei, CHEN Hangjun, YIN Junyi, FANG Xiangjun, WU Weijie, LIU Ruiling, HAN Yanchao, GAO Haiyan

2022, 34(8): 1734-1742. DOI: 10.3969/j.issn.1004-1524.2022.08.17

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This article explored the effects of different intestinal conditions and particle size on in vitro digestion of processed Carya cathayensis Sarg.(CCS), matrix degradation rate (MDI), digestibility of fat and protein, total phenols and antioxidant activity of processed CCS were analyzed under different bile salt concentration, intestinal pH values and pancreatic lipase concentrations according to the intestinal conditions of healthy adults and people with exocrine pancreatic insufficiency (EPI). The results showed particle size had the most significant effect on processed CCS in vitro digestion, and the MDI, antioxidant activity and digestibility of fat and protein of small particle processed CCS were higher than those of large particle processed CCS. The pH and bile salt were associated with fat digestion and antioxidant activity. When pH value was 7 and bile salt concentration was 10 mmol·L^-1, the fat digestion effect was better. The digestibility of fat and protein would be high when the concentration of pancreatic lipase was high. Therefore, people with EPI can take in the appropriate amount of prebiotics and trypsin preparation to improve the nutrient digestion and utilization rate of CCS products.

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Effects of blanching and drying on nutritional components and processing suitability of dried Promma microphylla Turcz leaves

XIN Xiaoting, LIU Daqun, ZHANG Jianming, ZHANG Chengcheng, YANG Guangting

2022, 34(8): 1743-1751. DOI: 10.3969/j.issn.1004-1524.2022.08.18

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In this paper, the effects of different processing methods on the nutritional components, antioxidant properties and the processing suitability of dried Promma microphylla Turcz leaves powder were studied. Blanching and different drying methods were used to treat Promma microphylla Turcz leaves, and they were divided into fresh (F), freeze-dried (FD), hot-air dried (HAD), fresh blanched (FB), blanched freeze-dried (BFD) and blanched hot-air dried (BHAD) group. The results showed that the leaves of FD group showed the best color, and the highest contents of total amino acids, reducing sugar, total phenol and total flavonoids were 36.16 mg·g^-1, 61.98 mg·g^-1, 6.31 mg·g^-1 and 106.56 mg·g^-1, respectively. The DPPH and FRAP values of FD, BFD and HAD were significantly higher than other treatments (P<0.05). Promma microphylla Turcz tofu was made from different treated Promma microphylla Turcz leaves dried powder. The BFD group tofu showed the best color with the lowest a^* value. In addition, BFD group tofu showed the shortest solidification time, highest yield, least exudate, and the best hardness, elasticity, cohesiveness, stickiness, chewiness, and sensory score of 83.33 ± 2.86. The BFD group retained the most nutritional efficacy and antioxidant capacity. As a result, blanched and freeze-dried treatment was the most suitable treatment for processing high-quality Promma microphylla Turcz leaves tofu.

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Optimization of DNA extraction method and DNA barcoding identification of walnut milk

HUANG Lingzhi, JIANG Guangze, DING Yanfei, ZHU Cheng

2022, 34(8): 1752-1761. DOI: 10.3969/j.issn.1004-1524.2022.08.19

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With the rapid development of milk beverage industry, adulteration of milk beverage was common, thus it is particularly important to accurately identify the true ingredients of walnut milk. Walnut milk is a deep-processed food with severe DNA damage. DNA extraction is the first step in DNA barcode identification of walnut milk. The purpose of this experiment was to optimize the DNA extraction method of walnut milk, and to establish an adulteration and fraud identification method based on psbA-trnH gene DNA barcoding. Taking 10 different brands of walnut milk as samples, three methods (static extraction, isopropanol precipitation and vacuum lyophilization) were used for pretreatment. Walnut milk DNA was extracted using two CTAB lysate precipitation methods and three kit methods (Kangwei Nuclean Plant Genomic DNA Kit, AxYGen AxyPrep Multisource Genomic DNA Miniprep Kit, and TianGen Deep Processing Food DNA Extraction Kit). Furthermore, CTAB and AxYGen kit was combined for DNA extraction. Using the extracted commercial walnut milk DNA as a template, the self-designed specific gene psbA-trnH-wal was used to identify whether the sample contained walnut components, and the fraud situation was determined. The common plant candidate gene psbA-trnH was selected to identify whether the sample contained other components, to determine adulteration. Vacuum freezing method was superior to the other two pretreatment methods. Walnut milk extracted DNA using CTAB and AxYGen kit combination method was in good purity, high yield and strong amplification ability, thus it was the best method for walnut milk DNA extraction. Amplification and comparison results showed that one walnut milk sample contained peanuts, which belonged to adulterated products. The vacuum freezing method and the combination of CTAB and AxYGen kit combination method were the optimized methods for DNA extraction from walnut milk. The combination of psbA-trnH-wal gene and psbA-trnH gene could quickly and accurately identify walnut milk and its adulterated fakes.

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Influence of polarized vector mode of polarization spectrum light state on polarized response effect of Locusta migratoria

LIU Qihang, GAO Xinguo, ZHOU Guotao, ZHOU Qiang

2022, 34(8): 1762-1771. DOI: 10.3969/j.issn.1004-1524.2022.08.20

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To clear the function effect of spectral polarized light state parameter on locusts polarized response effect, and obtain the technical characteristics of locusts polarization induction light field for manufacturing locusts polarization induction photo-source, utilizing locusts polarized light response test device, locusts response characteristics to partially and linearly polarized light with violet, blue spectrum, and the vector action mode of polarized light state was optimized to analyze the influence mechanism of polarized spectrum light state parameter on locusts polarized response effect, and discuss the reason for the sensitive change of locusts spectral polarized vector mode. Results showed that in partially polarized light, violet spectrum did not while blue spectrum caused the change of the sensitive vector, while in linearly polarized light, violet and blue spectrum all affected locusts sensitive vector mode, and under the same spectrum, locusts control induction effect, and polartaxic intensity caused by the vector directionality mode of partial polarized light were better than that of linearly polarized light, but under the same polarization vector light state, the function effect of violet spectrum was better than blue spectrum. Then, locusts polarized response effect were related to the heterogeneous initiation effect of polarized spectrum, and polarized light state, and locusts control induction effect caused by left and right 120° vector of linearly polarized violet light was the strongest (94.5%), while the function effect of locusts polartaxic aggregation degree stimulated by left 60° and right 150° vectors of partially polarized violet light was the strongest (68.5%). The effect of partial polarization vector orientation mode is related to the spectral photo-induced visual sensitivity polarization effect, and the higher the degree of polarization in the blue spectrum and the lower the illumination, the stronger the manipulation inducement of vector mode; the lower the degree of polarization in the purple spectrum and the stronger the illumination, the stronger the effect of vector mode on the degree of bias aggregation of locusts. Thus, the vector light alternating stimulation mode with different linear polarization and partial polarization can improve the sensitivity of locust's bias response.

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Design and experiment of swing intra-row weeding device for paddy field

TIAN Liang, CAO Chengmao, QIN Kuan, GE Jun, FANG Liangfei

2022, 34(8): 1772-1783. DOI: 10.3969/j.issn.1004-1524.2022.08.21

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Aiming at the problems of high seedling damage rate and low weeding rate of intra-row weeding device in paddy field, a swing intra-row weeding device for paddy field was designed. The overall structure and working principle of weeding device were illustrated and analyzed. Theoretical analysis and structural design of eccentric mechanism and weeding spring tine were carried out. Combined with the reciprocating opening and closing movement of spring tine and rice planting agronomy, the movement process of weeding spring tine and the working mechanism of seedling avoidance system were analyzed. Based on ADAMS software, by taking the machine driving speed and eccentric rotational speed as test factors, coverage rate and intrusion rate as evaluation indexes, the virtual simulation test was carried out via two factor five level quadratic regression orthogonal rotation combination method. The simulation results revealed that the machine driving speed and eccentric rotation speed had significant (P<0.01) impact on the coverage rate and intrusion rate, and the machine driving speed was the main factor affecting the working performance of the weeding device. When the machine driving speed was 266.12 mm·s^-1 and the eccentric rotational speed was 3.97 r·s^-1, the coverage rate was 85.70% and the intrusion rate was 5.62% in simulation. The field test results revealed that when the machine driving speed was 270 mm·s^-1 and the eccentric rotational speed was 4 r·s^-1, the weeding rate of the weeding device was 82.5% and the injury rate was 5.1%, which was roughly consistent with the simulation results. Thus, the designed weeding device could meet the requirement of weeding agronomy in paddy field.

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Calculation and analysis of contribution rate of technological progress of agriculture in Zhejiang Province, China: based on stochastic frontier analysis

MAO Xiaohong, FU Linlin, ZHANG Liu, WANG Jin, MAO Xiaobao

2022, 34(8): 1784-1795. DOI: 10.3969/j.issn.1004-1524.2022.08.22

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In the present study, based on the county-level panel data, stochastic frontier analysis was introduced to calculate the contribution rate of technological progress of agriculture in Zhejiang in 2014-2019. It was shown that the growth of agricultural output in Zhejiang in 2014-2019 was mainly driven by the improvement of technological progress rate. In 2019, the contribution rate of technological progress of agriculture in Zhejiang reached 64.30%. In 2014-2019, the narrow technological progress rate showed a steady increasing trend, which was mainly driven by neutral technological progress. Changes in the agricultural industry structure affected the contribution rate of technological progress of agriculture via its influence on the application of related technologies and the scale of production and operation. Three agricultural development types were divided in Zhejiang as plain type, coastal type and mountain type. Among them capital and land had bigger impact on the mountain type, while labor had bigger impact on the plain type. Costal type showed the highest change rate of technological efficiency.

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Driving effect and influence path of “agricultural makers” in rural development of Zhejiang Province, China

FU Jiayi, TONG Lei, SU Fei, ZHENG Ke, LI Hailin, CAO Yirong, SU Na

2022, 34(8): 1796-1805. DOI: 10.3969/j.issn.1004-1524.2022.08.23

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In the context of rural revitalization, Zhejiang Province, China was selected as the case area in the present study. Based on the filed survey, the driving effect and influence path of “agricultural makers” in rural development were explored via factorial analysis etc. It was shown that “agricultural makers” played obvious driving effect in rural development, and the effect was concentrated in the aspects of attracting investment and increasing the number of college students returning hometown. According to the driving mode in rural development, “agricultural makers” could be divided into 4 types, namely, “brand creation type” “innovative technology type” “innovative organization type” and “entrepreneurial growth type”, and the driving effect of the above 4 types decreased in sequence. “Agricultural makers” changed the financial capital, material capital, social capital and human capital in the process of innovation and entrepreneurship. During the rural development, the contribution of these four kinds of capitals decreased in sequence. There was a clear path dependence within financial capital, material capital, social capital and human capital. Among them, financial capital was the basis, and played a key role in rural development, while human capital was the most indirect one. Finally, corresponding optimization suggestions were proposed to accurately improve the driving effect of “agricultural makers” in rural development.

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Application and policy regulation of CRISPR/Cas9 gene editing technology in plants

OU Chun, ZHANG Min, DING Lin, YAO Xiamei, WANG Zelu, PENG Cheng, XU Junfeng

2022, 34(8): 1806-1814. DOI: 10.3969/j.issn.1004-1524.2022.08.24

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Gene editing technology is a technology that making precise modification of DNA sequence at the genome level, so as to promote targeted modification of the genome sequence. With the rapid development of CRISPR/Cas9 technology in recent years, gene editing technology plays an increasingly important role in crop breeding. The paper reviewed the development of gene editing technology and the working principle of CRISPR/Cas9, analysed the limitations of CRISPR/Cas9 and put forward improvement methods. In addition, this paper focused on the establishment of the plant CRISPR/Cas9 gene editing system, its application in plant trait improvement, and the application cases that were finally committed to the commercialization of gene editing products. At the same time, it also analysed the regulatory policies and attitudes of gene editing in five representative countries/regions, including the United States, the European Union, Britain, Japan and China, in order to provide reference for the establishment of scientific regulatory frame work in China and promote the industrial application of CRISPR/Cas9 in China or even the world.

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