Cloning and target gene screening of miR397 in potato

doi:10.3969/j.issn.1004-1524.2022.06.05

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (6): 1141-1151.DOI: 10.3969/j.issn.1004-1524.2022.06.05

• Crop Science • Previous Articles Next Articles

Cloning and target gene screening of miR397 in potato

LI Wenxiang¹(), WANG Fang¹^,²^,³^,⁴^,⁵^,⁶^,^*(), WANG Jian¹^,²^,³^,⁴^,⁵^,⁶

1. College of Agriculture and Animal HUsbandry, Qinghai University, Xining 810016, China
2. Qinghai Academy of Agriculture and Forestry Sciences, Xining 810016, China
3. Key Laboratory of Qinghai-Tibetan Plateau Biotechnology of Ministry of Education, Xining 810016, China
4. Key Laboratory of Potato Breeding of Qinghai, Xining 810099, China
5. Engineering Research Center of Potato in Northwest Region, Ministry of Education, Xining 810016, China
6. State key Laboratory of Plateau Ecology and Agriculture, Xining 810016, China

Received:2022-02-22 Online:2022-06-25 Published:2022-06-30
Contact: WANG Fang

Abstract

Abstract:

miR397 is widely involved in plant stress response. In this study, the precursor sequence of miR397 was isolated from potato leaves, the StmiR397 was cloned and its potential target genes were predicted. The qRT-PCR was used to analyze the expression patterns of different low-temperature phenotypic materials, the results showed: the length of precursor sequence of potato miR397 was 81 bp, which could form stable stem-loop structure, and the mature sequence was located at the 5' end. The upstream regulatory region contained various stress response elements such as temperature response. Under low temperature treatment, the expression of StmiR397 was up-regulated in the leaves of the low-temperature resistant material DR-2, and the expression showed no difference in the roots, while the expression of StmiR397 showed no difference in the leaves of the low-temperature sensitive material Favorite, and the expression was up-regulated in the roots. According to the expression patterns analyses of the 10 predicted target genes, oxidoreductase-like domain-containing protein gene, zinc knuckle (CCHC-type) family protein gene of the reverse inhibitory target genes and the positive activation target genes 6-phosphate 1-phosphotransferase subunit alpha gene and auxin-induced beta-glucosidase gene were initially screened. This study provides a certain reference for further research on the regulatory network of StmiR397 and target genes.

Key words: potato, low temperature stress, target gene

CLC Number:

S532

LI Wenxiang, WANG Fang, WANG Jian. Cloning and target gene screening of miR397 in potato[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1141-1151.

Figures/Tables 9

References 33

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引物名称 Primer name	引物序列 Primer sequence（5’→3’）
StmiR397-F	ATTGAGTGCAGCGTTGATGAC
StmiR397-R	TGATTGAGTGTAGCGTTGATGAAG
LAC7-F	CATTTGGATGTTCACTTGCCTTGGG
LAC7-R	CTGGTGGAGGAGGAGGTAGCATAG
M1C3Q2-F	ACAAGTCATTACTACTGCCGCTTCC
M1C3Q2-R	TAGTCCTGCTGCTCCTCCGTTATAC
ICE-F	GATGGACAGGGCTTCAATCTTAGGG
ICE-R	CGGCGTGGACTCCAGTTCATTATG
M1BIR1-F	TCGGCTACATTCACCAACAACACC
M1BIR1-R	ACCGCTGAAAGGATTGCTACACTG
M0ZH35-F	CGGTGGACAGTGATGGGACATTC
M0ZH35-R	AGCCTAAACGAGAACCAGCAAGATC
M1BHR0-F	GATCCAGATAACCGCCGCAAGAG
M1BHR0-R	TGCCCTTCCTGTCCACAATGTTTAC
LAC3-F LAC3-R	CACCACTACCACTGCCATACTTGAG TGGCTGTATCATTGAAGGCTGGAAG
M1CV13-F		TACCGAGCATCCAGCGAAGAGG
M1CV13-R		GAACCACCCAGAAAACCAAGCAAAG
M1C9X3-F		GACCATCCAGCGACTCTTCACTTG
M1C9X3-R		TGCCTTTCCAGTGCTTGTAGTTCTC
M1AZQ5-F		AGGGAAATGGGAGAGGACAAGAGAG
M1AZQ5-R		CTGTATTCGCTGGTCTGCTGATCTG
M0ZW04-F		AACCAATCTTCCTCATCGGCAACC
M0ZW04-R		GCAGCGAAATTACGGCTTGATGG
miR397-F		CGCGATTGAGTGCAGCGTT
StA F		AGATGCTTACGCTGGATGGAATGC
StA R		TTCCGGTGTGGTTGGATTCTGTTC

引物名称 Primer name	引物序列 Primer sequence（5’→3’）
StmiR397-F	ATTGAGTGCAGCGTTGATGAC
StmiR397-R	TGATTGAGTGTAGCGTTGATGAAG
LAC7-F	CATTTGGATGTTCACTTGCCTTGGG
LAC7-R	CTGGTGGAGGAGGAGGTAGCATAG
M1C3Q2-F	ACAAGTCATTACTACTGCCGCTTCC
M1C3Q2-R	TAGTCCTGCTGCTCCTCCGTTATAC
ICE-F	GATGGACAGGGCTTCAATCTTAGGG
ICE-R	CGGCGTGGACTCCAGTTCATTATG
M1BIR1-F	TCGGCTACATTCACCAACAACACC
M1BIR1-R	ACCGCTGAAAGGATTGCTACACTG
M0ZH35-F	CGGTGGACAGTGATGGGACATTC
M0ZH35-R	AGCCTAAACGAGAACCAGCAAGATC
M1BHR0-F	GATCCAGATAACCGCCGCAAGAG
M1BHR0-R	TGCCCTTCCTGTCCACAATGTTTAC
LAC3-F LAC3-R	CACCACTACCACTGCCATACTTGAG TGGCTGTATCATTGAAGGCTGGAAG
M1CV13-F		TACCGAGCATCCAGCGAAGAGG
M1CV13-R		GAACCACCCAGAAAACCAAGCAAAG
M1C9X3-F		GACCATCCAGCGACTCTTCACTTG
M1C9X3-R		TGCCTTTCCAGTGCTTGTAGTTCTC
M1AZQ5-F		AGGGAAATGGGAGAGGACAAGAGAG
M1AZQ5-R		CTGTATTCGCTGGTCTGCTGATCTG
M0ZW04-F		AACCAATCTTCCTCATCGGCAACC
M0ZW04-R		GCAGCGAAATTACGGCTTGATGG
miR397-F		CGCGATTGAGTGCAGCGTT
StA F		AGATGCTTACGCTGGATGGAATGC
StA R		TTCCGGTGTGGTTGGATTCTGTTC

元件Element	序列 Sequence	功能Function
TGACG-motif	TGACG	参与茉莉酸响应的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness
TCA-element	CCATCTTTTT	参与水杨酸响应的顺式作用元件 cis-acting element involved in salicylic acid responsiveness
TATC-box	TATCCCA	参与赤霉素响应的顺式作用元件 cis-acting element involved in gibberellin-responsiveness
ABRE	ACGTG	参与脱落酸响应的顺式作用元件 cis-acting element involved in the abscisic acid responsiveness
TATA-box	ATATAT	核心启动子元件Core promoter element
TATA-box	TATA	核心启动子元件Core promoter element
TATA-box	TATAAA	核心启动子元件Core promoter element
TATA-box	TATAA	核心启动子元件Core promoter element
CGTCA-motif	CGTCA	参与茉莉酸响应的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness
P-box	CCTTTTG	赤霉素响应元件Gibberellin-responsive element
CAAT-box	CAAAT	启动子和增强子区域的常见顺式作用元件 Common cis-acting element in promoter and enhancer regions
MRE	AACCTAA	参与光响应MYB结合位点MYB binding site involved in light responsiveness
3-AF1 binding site	TAAGAGAGGAA	光响应元件Light responsive element
TCT-motif	TCTTAC	光响应元件的一部分Part of a light responsive element
I-box	gGATAAGGTG	光响应元件的一部分Part of a light responsive element
GT1-motif	GGTTAA	光响应元件Light responsive element
ARE	AAACCA	厌氧诱导所必需的顺式作用调节元件 cis-acting regulatory element essential for the anaerobic induction
G-Box	CACGTT	参与光响应的顺式作用调节元件 cis-acting regulatory element involved in light responsiveness
TC-rich repeats	GTTTTCTTAC	参与防御和应激反应的顺式作用元件 cis-acting element involved in defense and stress responsiveness
CCAAT-box	CAACGG	MYBHv1结合位点MYBHv1 binding site
HSE	CTCC	参与温度响应的顺式作用元件 cis-acting element involved in temperature responsiveness

元件Element	序列 Sequence	功能Function
TGACG-motif	TGACG	参与茉莉酸响应的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness
TCA-element	CCATCTTTTT	参与水杨酸响应的顺式作用元件 cis-acting element involved in salicylic acid responsiveness
TATC-box	TATCCCA	参与赤霉素响应的顺式作用元件 cis-acting element involved in gibberellin-responsiveness
ABRE	ACGTG	参与脱落酸响应的顺式作用元件 cis-acting element involved in the abscisic acid responsiveness
TATA-box	ATATAT	核心启动子元件Core promoter element
TATA-box	TATA	核心启动子元件Core promoter element
TATA-box	TATAAA	核心启动子元件Core promoter element
TATA-box	TATAA	核心启动子元件Core promoter element
CGTCA-motif	CGTCA	参与茉莉酸响应的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness
P-box	CCTTTTG	赤霉素响应元件Gibberellin-responsive element
CAAT-box	CAAAT	启动子和增强子区域的常见顺式作用元件 Common cis-acting element in promoter and enhancer regions
MRE	AACCTAA	参与光响应MYB结合位点MYB binding site involved in light responsiveness
3-AF1 binding site	TAAGAGAGGAA	光响应元件Light responsive element
TCT-motif	TCTTAC	光响应元件的一部分Part of a light responsive element
I-box	gGATAAGGTG	光响应元件的一部分Part of a light responsive element
GT1-motif	GGTTAA	光响应元件Light responsive element
ARE	AAACCA	厌氧诱导所必需的顺式作用调节元件 cis-acting regulatory element essential for the anaerobic induction
G-Box	CACGTT	参与光响应的顺式作用调节元件 cis-acting regulatory element involved in light responsiveness
TC-rich repeats	GTTTTCTTAC	参与防御和应激反应的顺式作用元件 cis-acting element involved in defense and stress responsiveness
CCAAT-box	CAACGG	MYBHv1结合位点MYBHv1 binding site
HSE	CTCC	参与温度响应的顺式作用元件 cis-acting element involved in temperature responsiveness

名称Name	基因描述Gene description	靶向mRNA ID Target mRNA	起始位置Target start	终止位置Target end	得分Score	靶向比对序列Target aligned Fragment	碱基配对Match	miRNA比对序列miRNA aligned fragment	功能Function
LAC7	漆酶7 Laccase-7-like	Soltu.DM.07G016640.1	676	696	1	AUCAUCAACGCUGCACUCAAU	::::::::::::::::::::	CAGUAGUUGCGACGUGAGUUA	催化活性、结合Catalytic activity, binding
LAC3	漆酶3 Laccase-3-like	Soltu.DM.06G032550.1	433	453	1	GUGAUCAACGCUGCACUCAAU	:: ::::::::::::::::::	CAGUAGUUGCGACGUGAGUUA	催化活性、结合Catalytic activity, binding
M1BIR1（PPR）	含五肽重复序列的蛋白质 Pentatricopeptide repeat-containing protein	Soltu.DM.09G030600.1	1021	1040	6	GUCAUAACUGCUGCA-UCAAA	::::: :.:::::: ::::	CAGUAGUUGCGACGUGAGUUA	生物过程Biological process
M0ZH35 （APs1）	天冬氨酸蛋白酶1 Aspartic proteinase 1	Soltu.DM.01G035170.1	674	694	6	GUAAGAAAUGCUACACUCAAU	:: : ::.::: ::::::::	CAGUAGUUGCGACGUGAGUUA	天冬氨酸型内肽酶活性、水解酶活性、非生物刺激响应Aspartic endopeptidase activity, hydrolase activity, abiotic stimulation response
M1BHR0（CCHC）	锌指家族蛋白 Zinc knuckle (CCHC-type) family protein	Soltu.DM.05G006320.2	18	37	4	GUCAUCAAUGUUGC-CUCAAU	::::::::.:.::: ::::::	CAGUAGUUGCGACGUGAGUUA	mRNA结合、翻译调节活性、锌离子结合、核酸结合mRNA binding, translation regulatory activity, zinc ion binding, nucleic acid binding
M0ZW04 (Olp)	氧化还原酶结构域蛋白 Oxidoreductase-like domain-containing protein	Soltu.DM.02G024460.1	304	323	4.5	GUAAUUU-CGC UGCACUCAAA	:: ::. ::::::::::::	CAGUAGUUGCGACGUGAGUUA	氧化还原酶活性Oxidoreductase activity
M1AZQ5 (CTC7)	含五肽重复序列的蛋白质 Pentatricopeptide repeat-containing protein	Soltu.DM.08G011800.1	455	476	5	GUCAUCAACGUCUGCAGUUAAU	:::::::::: ::::: :.:::	CAGUAGUUGCGACGUGAGUUA	蛋白质结合、mRNA结合Protein binding, mRNA binding
M1C9X3 （MIS12）	动力蛋白复合物MIS12 homologue	Soltu.DM.03G028560.1	177	197	5	UUCAUCAACGAGGCACUCAAU	::::::::: :::::::::	CAGUAGUUGCGACGUGAGUUA	细胞过程、细胞成分组织、细胞分裂Cell process, cell composition, tissue, cell division
M1CV13 （PFP）	磷酸果糖激酶家族蛋白6-phosphate 1-phosphotransferase subunit alpha	Soltu.DM.12G004610.1	1839	1859	6	GUCCUGAAAGCUGCACUUAGU	::: : :: ::::::::.:.:	CAGUAGUUGCGACGUGAGUUA	6-磷酸果糖激酶活性、 ATP结合、激酶活性、光合作用Fructokinase-6-phosphate activity, ATP binding, kinase activity, photosynthesis
M1C3Q2（GHs1）	生长素诱导的β-葡萄糖苷酶 Auxin-induced beta-glucosidase	Soltu.DM.11G017090.1	330	349	6	GUGAACAAUGCUGCAC-CAGU	:: : :::.::::::: ::.:	CAGUAGUUGCGACGUGAGUUA	水解酶活性、分解代谢过程、碳水化合物代谢过程Hydrolase activity, catabolic process, carbohydrate metabolism process

Cloning and target gene screening of miR397 in potato

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[1]	LI Xiaolan, ZHANG Rui, HAO Lanlan, WANG Hong. Bioinformatics analysis of peach NAC gene family and its expression characteristics in response to low temperature stress [J]. Acta Agriculturae Zhejiangensis, 2022, 34(4): 766-780.
[2]	DING Yanling, WANG Pengfei, YANG Chaoyun, ZHOU Xiaonan, ZHAO Zhiyan, ZHANG Yanfeng, SHI Yuan- gang, KANG Xiaolong. Prediction of target genes and tissue expression analysis of miR-144 in cattle [J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 471-479.
[3]	YUAN Wenya, KANG Yichen, YANG Xinyu, ZHNAG Ruyan, ZHOU Chuntao, WANG Yong, CHEN Xipeng, YU Huifang, QIN Shuhao. Effects of rhizosphere soil extract of Qingshui alfalfa (Medicago sativa L.) on enzyme activities and microbial communities in rhizosphere soil of continuous cropping potato [J]. Acta Agriculturae Zhejiangensis, 2022, 34(2): 240-247.
[4]	YE Jing, YANG Yuanling, SHI Qingqiu, WU Longfei, SONG Guotao. Bioinformatics analysis and function prediction of miR172 gene family in Eucommia ulmoides [J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 70-78.
[5]	HUANG Changbing, CHENG Peilei, YANG Shaozong, ZHANG Huanchao, JIANG Zhengzhi, JIN Limin. Transcriptome analysis of Hemerocallis fulva under low temperature stress [J]. Acta Agriculturae Zhejiangensis, 2021, 33(8): 1445-1460.
[6]	CHEN Wen, ZHANG Weiwei, SHAO Shuli, FU Xuepeng, HUANG Xin, LI Tie. Expression of miR-423-5p in bovine muscle and predicted target genes [J]. Acta Agriculturae Zhejiangensis, 2021, 33(5): 785-793.
[7]	WANG Haiyi, ZHANG Zhaoguo, IBRAHIM Issa, XIE Kaiting, Wael EL-KOLALY, CAO Qinzhou. Design and experiment of small-sized potato harvester suitable for hilly and mountainous areas [J]. Acta Agriculturae Zhejiangensis, 2021, 33(4): 724-738.
[8]	XIE Ziyu, WANG Ke’er, ZHAO Wenliang, WEN Zuhui, CHEN Linrun, XU Lishan. Nutritional components and bioactivities of sweet potatoes with different flesh colors [J]. Acta Agriculturae Zhejiangensis, 2021, 33(2): 183-192.
[9]	ZHAO Lin, YE Xiafang, DONG Wei, SHI Jiang, LUO Letan, LUO Guoquan. Changes of nutritional quality and starch properties of different types of sweet potato roots during storage [J]. Acta Agriculturae Zhejiangensis, 2021, 33(12): 2224-2233.
[10]	LIANG Liqin, YANG Rui, GAO Gang. Bioinformatics analysis of StUOXs gene family in potato [J]. , 2020, 32(9): 1523-1532.
[11]	WANG Wei, GUN Shuangbao, WANG Pengfei, HUANG Xiaoyu, XIE Kaihui, LUO Ruirui, GAO Xiaoli, ZHANG Bo, YAN Zunqiang, YANG Qiaoli, MA Yanping. Tissue expression and significant target genes analysis of swine miR-204 [J]. , 2020, 32(9): 1564-1573.
[12]	XU Jianmin, SHI Hedi, SHI Peihua, ZHANG Zeyang, XU Zhigang. Comparison of fitting models of light response curve of potato under different light quality [J]. , 2020, 32(5): 753-761.
[13]	LIU Ziying, YUAN Bin, XIAO Huamei, WU Yongfei, LIU Xiaolin, HU Xiangfei. Screening, identification of Phytophthora infestans and its antagonistic bacterial strain [J]. , 2020, 32(5): 840-848.
[14]	WU Weicheng, DAI Jianbo, CAO Yan, XIA Qile, CHEN Jianbing, MENG Xianghe. Effects of physical modification on content, polysaccharide composition and structure of dietary fiber in sweet potato peels [J]. , 2020, 32(3): 490-498.
[15]	HAN Tiqian, LIU Zhen, LIU Yuhui, ZHANG Xiaojing, WANG Li, ZHANG Junlian. Effects of reduced chemical nitrogen and organic manure substitution on potato root morphology and yield [J]. Acta Agriculturae Zhejiangensis, 2020, 32(12): 2111-2118.