热处理对转基因秸秆中重组蛋白和重组DNA的降解作用

doi:10.3969/j.issn.1004-1524.20240332

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (9): 2079-2088.DOI: 10.3969/j.issn.1004-1524.20240332

热处理对转基因秸秆中重组蛋白和重组DNA的降解作用

颜晶莹(), 倪亮, 沈星宇, 李玉

浙江大学农业试验站,浙江杭州 310058

收稿日期:2024-04-11 出版日期:2024-09-25 发布日期:2024-09-30
作者简介:颜晶莹(1988—),女,湖南长沙人,博士,讲师,主要从事转基因快速检测研究。E-mail: 463278@zju.edu.cn
基金资助:
浙江大学实验技术研究项目(SYB202138);国家自然科学基金(32000195)

Effect of heat treatment on the degradation of recombinant protein and recombinant DNA in transgenic straws

YAN Jingying(), NI Liang, SHEN Xingyu, LI Yu

Agricultural Experimental Station, Zhejiang University, Hangzhou 310058, China

Received:2024-04-11 Online:2024-09-25 Published:2024-09-30

摘要/Abstract

摘要：

随着转基因作物种植面积的不断扩大,如何高效处理转基因秸秆成为了一个重要的科学问题。未经处理的转基因秸秆中的重组蛋白和重组DNA可以在土壤中存在很长时间,并对土壤生物多样性产生潜在负面影响。因此寻找一个既节约成本又对环境无害的秸秆处理方法非常重要。高温处理是降解转基因秸秆重组蛋白和重组DNA的有效手段,但目前对处理温度和处理时间的选择还缺乏系统的研究。本研究通过试纸条、聚合酶链反应(PCR)等方法检测不同温度和时间处理的转基因作物秸秆中重组蛋白和重组DNA的水平。结果表明,转基因大豆、棉花、玉米、水稻的秸秆在50 ℃处理3 h后,其体内的抗除草剂蛋白草胺膦乙酰转移酶(phosphinothricin acetyltransferase, PAT)等重组蛋白基本降解;但相同温度下,重组DNA的降解则需要4 d时间;提高处理温度可以在一定程度上缩短重组蛋白和重组DNA降解所需的时间。50 ℃处理4 d的条件在实际生产中通过堆肥就能实现。本研究从分子生物学的角度为转基因秸秆处理提供了依据。

关键词: 转基因秸秆, 重组蛋白, 重组DNA, 草胺膦乙酰转移酶(PAT), 5-烯醇式丙酮莽草酸-3-磷酸合酶基因(Cp4-EPSPS), Bt基因

Abstract:

With the continuous expansion of transgenic crop planting area, how to efficiently deal with the transgenic straw has become an important scientific issue. The recombinant protein and recombinant DNA in untreated transgenic straw can persist in the soil for a long time and have a potentially negative impact on soil biodiversity. Therefore, it is of importance to find a cost-saving and environmentally friendly straw treatment method. High temperature treatment is an effective method to degrade the recombinant protein and recombinant DNA of transgenic straw, but the treatment temperature and treatment time have not been systematically studied at present. This study used the test strips and polymerase chain reaction (PCR) to detect the levels of recombinant protein and recombinant DNA in the transgenic straw under different temperature and time treatments, and found that phosphinothricin acetyltransferase (PAT) and other recombinant proteins in the straw of transgenic soybeans, cotton, maize and rice were almost degraded after being treated at 50 ℃ for 3 h. However, at the same temperature, the degradation of recombinant DNA took 4 d. The degradation time of recombinant proteins and recombinant DNA could be shorted by increasing the temperature. These combinations of temperature and time, particularly the treatment condition of 50 ℃ for 4 d, could be achieved by composting in agricultural production. Therefore, this study provided a theoretical basis for the treatment of transgenic straw from the perspective of molecular biology.

Key words: transgenic straw, recombinant protein, recombinant DNA, phosphinothricin acetyltransferase (PAT), CP4 5-enolpyruvyl-shikimate-3-phosphate synthase gene (Cp4-EPSPS), Bacillus thuringiensis (Bt) gene

中图分类号:

S141.4

颜晶莹, 倪亮, 沈星宇, 李玉. 热处理对转基因秸秆中重组蛋白和重组DNA的降解作用[J]. 浙江农业学报, 2024, 36(9): 2079-2088.

YAN Jingying, NI Liang, SHEN Xingyu, LI Yu. Effect of heat treatment on the degradation of recombinant protein and recombinant DNA in transgenic straws[J]. Acta Agriculturae Zhejiangensis, 2024, 36(9): 2079-2088.

图/表 7

表1 引物信息

Table 1 Primer information

基因 Gene	正向引物序列 Positive primer sequence (5’→3’)	反向引物序列 Reverse primer sequence(5’→3’)	产物长度 Product length/bp
CaMV35S	GATAGTGGAAAAGGAAGGTGGC	GAAGGGTCTTGCGAAGGATAG	230
STOP1-GFP	CTCTGTTCCAGGGACACACG	GTCCATGCCGTGAGTGATCC	1 100

图1 不同温度和时间的热处理对转基因大豆秸秆重组蛋白降解的影响 A,不同温度和时间对转基因大豆秸秆中PAT蛋白降解的影响;B,不同温度对转基因大豆秸秆PAT蛋白降解的影响;C,不同温度对转基因大豆秸秆中Cp4-EPSPS蛋白降解的影响;D,不同温度对转基因大豆秸秆中Bt蛋白降解的影响。CK为常温对照组;N为阴性对照组,即试纸条检测非转基因大豆秸秆的结果。蓝色箭头为质控线,红色箭头为检测线。

Fig.1 Effect of heat treatments with different temperature and time on the recombinant protein degradation of transgenic soybean straw A, Effects of different temperature and time treatments on PAT degradation of transgenic soybean straw; B, Effect of different temperature treatments on PAT degradation of transgenic soybean straw; C, Effect of different temperature treatments on Cp4-EPSPS degradation of transgenic soybean straw; D, Effect of different temperature treatments on Bt degradation of transgenic soybean straw. CK indicated treatment under normal temperature; N indicated negative control with detection of wild-type soybean straw. The blue arrow indicated quality control line, and the red arrow indicated detection line.

图2 不同温度的热处理对转基因拟南芥GFP融合表达蛋白稳定性的影响 A,常温下转基因拟南芥根尖的GFP融合表达蛋白;B、C、D,不同温度处理后转基因拟南芥根尖的GFP融合表达蛋白。

Fig.2 Effect of different heat treatments on GFP fusion protein stability in transgenic Arabidopsis A, GFP fusion protein in transgenic Arabidopsis root tip under normal temperature; B, C and D, GFP fusion protein in transgenic Arabidopsis root tip under different temperatures.

图3 不同温度的热处理对转基因拟南芥GUS活性的影响 A,常温下转基因拟南芥叶片的GUS染色;B、C、D,不同温度处理后转基因拟南芥叶片的GUS染色。

Fig.3 Effect of different heat treatments on GUS activity of transgenic Arabidopsis A, GUS staining of transgenic Arabidopsis leaf under normal temperature; B, C and D, GUS staining of transgenic Arabidopsis leaf under different temperatures.

图4 50 ℃处理3 h对不同转基因作物新鲜秸秆中重组蛋白降解的影响 A,转PAT基因作物秸秆;B,转Cp4-EPSPS基因作物秸秆;C,转Bt基因作物秸秆。N指阴性对照,即非转基因作物;CK为常温对照;L为叶;S为茎。蓝色箭头为质控线,红色箭头为检测线。下同。

Fig.4 Effects of 50 ℃ treatment for 3 h on the degradation of recombinant proteins in fresh straw of different transgenic crops A, PAT transgenic crop straw; B, Cp4-EPSPS transgenic crop straw; C, Bt transgenic crop straw. N indicated negative control with detection of wild type crops; CK indicated treatment under normal temperature; L indicated leaf; S indicated stem. The blue arrow indicated quality control line, and the red arrow indicated detection line. The same as below.

图5 50℃处理3 h对不同转基因作物成熟秸秆重组蛋白降解的影响

Fig.5 Effects of 50 ℃ treatment for 3 h on the degradation of recombinant proteins in mature straw of different transgenic crops

图6 不同温度和时间的热处理对转基因大豆秸秆DNA稳定性的影响 A,不同温度和时间热处理对转基因大豆秸秆中GFP融合基因稳定性的影响;B,不同温度和时间热处理对转基因大豆秸秆中CaMV35S序列稳定性的影响;GFP融合片段大小为1 100 bp,CaMV35S片段大小为230 bp;M,DL 2 000 marker;1~3号泳道(N)为阴性对照,对应样品为非转基因大豆秸秆;4~6号泳道(CK)对应样品为常温处理的转基因大豆秸秆;7~9和16~18号泳道对应样品为50 ℃处理的转基因大豆秸秆;10~12和19~21号泳道对应样品为70 ℃处理的转基因大豆秸秆;13~15和22~24号泳道对应样品为90 ℃处理的转基因大豆秸秆;*表示非特异性条带。C,50 ℃处理不同时间对转基因大豆秸秆中CaMV35S序列稳定性的影响;1~3号泳道对应样品为常温下的新鲜转基因大豆秸秆,4~6号泳道对应样品为室温下放置3 d的转基因大豆秸秆,7~21号泳道对应样品为50℃处理不同时间的转基因大豆秸秆,22~24号泳道对应样品为ddH2O。

Fig.6 Effects of heat treatment at different temperatures and time on the stability of transgenic soybean straw DNA A, Effects of heat treatment at different temperatures and time on the stability of GFP fusion gene in transgenic soybean straw; B, Effects of heat treatment at different temperatures and time on the stability of CaMV35S sequence in transgenic soybean straw; The length of GFP fusion fragment was 1 100 bp, the length of CaMV35S fragment was 230 bp; M, DL 2 000 marker; Lanes 1-3 (N) indicated negative control of non-transgenic soybean straw, lanes 4-6 indicated transgenic soybean straw treated at normal temperature, lanes 7-9 and 16-18 indicated transgenic soybean straw treated at 50 ℃, lanes 10-12 and 19-21 indicated transgenic soybean straw treated at 70 ℃, lanes 13-15 and 22-24 indicated transgenic soybean straw treated at 90 ℃; * indicated non-specific band. C, Effects of different time of 50 ℃ treatment on the stability of CaMV35S sequence in transgenic soybean straw; Lanes 1-3 indicated fresh transgenic soybean straw, lanes 4-6 indicated transgenic soybean straw treated at normal temperature for 3 d, lanes 7-21 indicated transgenic soybean straw treated at 50 ℃ for different time, the samples in lanes 22-24 were ddH2O.

参考文献 28

[1]	美国转基因作物种植比例攀升[J]. 农村新技术, 2020, 9: 39.
	The proportion of genetically modified crops planted in the United States is on the rise[J]. New Rural Technology, 2020, 9: 39. (in Chinese).
[2]	芦宝琳. 转基因作物的发展[J]. 中国甜菜糖业, 2021(4): 38-41.
	LU B L. Development of genetically modified crops[J]. China Beet & Sugar, 2021(4): 38-41. (in Chinese)
[3]	杨晓然, 刘靖, 姚淑军, 等. 3种不同抗除草剂转基因大豆对田间节肢动物多样性的影响[J]. 生态与农村环境学报, 2023, 39(4): 504-510.
	YANG X R, LIU J, YAO S J, et al. Effects of three different herbicide-resistant transgenic soybeans on arthropod diversity in field[J]. Journal of Ecology and Rural Environment, 2023, 39(4): 504-510. (in Chinese with English abstract)
[4]	JIANG Z L, ZHOU L, WANG B F, et al. Construction of a novel degradation model of Bacillus thuringiensis protein in soil and its application in estimation of the degradation dynamics of bt-Cry1Ah protein[J]. Frontiers in Plant Science, 2022, 13: 875020.
[5]	MANDAL A, SARKAR B, OWENS G, et al. Impact of genetically modified crops on rhizosphere microorganisms and processes: a review focusing on Bt cotton[J]. Applied Soil Ecology, 2020, 148: 103492.
[6]	YURCHAK V, LESLIE A W, DIVELY G P, et al. Degradation of transgenic Bacillus thuringiensis proteins in corn tissue in response to post-harvest management practices[J]. Transgenic Research, 2021, 30(6): 851-865.
[7]	DENG J X, WANG Y M, YANG F Y, et al. Persistence of insecticidal Cry toxins in Bt rice residues under field conditions estimated by biological and immunological assays[J]. Science of the Total Environment, 2019, 679: 45-51.
[8]	ZHANG M J, FENG M C, XIAO L J, et al. Persistence of Cry1Ac protein from transgenic bt cotton cultivation and residue returning in fields and its effect on functional diversity of soil microbial communities[J]. Pedosphere, 2019, 29(1): 114-122.
[9]	吕箫, 冯远娇, 王晓宜, 等. Bt作物秸秆还田对土壤生态系统的影响研究进展[J]. 生态科学, 2019, 38(3): 235-240.
	LÜX, FENG Y J, WANG X Y, et al. Research progress on the effects of Bt crops straw return on soil ecosystem[J]. Ecological Science, 2019, 38(3): 235-240. (in Chinese with English abstract)
[10]	LI Y J, WANG C, GE L, et al. Environmental behaviors of Bacillus thuringiensis(Bt) insecticidal proteins and their effects on microbial ecology[J]. Plants, 2022, 11(9): 1212.
[11]	GE L, SONG L L, WANG L Y, et al. Evaluating response mechanisms of soil microbiomes and metabolomes to Bt toxin additions[J]. Journal of Hazardous Materials, 2023, 448: 130904.
[12]	RIAZ MARRAL M W, KHAN M B, AHMAD F, et al. The influence of transgenic (Bt) and non-transgenic (non-Bt) cotton mulches on weed dynamics, soil properties and productivity of different winter crops[J]. PLoS One, 2020, 15(9): e0238716.
[13]	SONG Y Y, LIU R Y, WANG M F, et al. Effects of transgenic Bt rice lines with single Cry1Ab and fused Cry1Ab/Cry1Ac on the abundance dynamics and community diversity of soil mites[J]. Archives of Agronomy and Soil Science, 2020, 66(5): 586-599.
[14]	JIAN Z H, ZENG L, XU T J, et al. Antibiotic resistance genes in bacteria: occurrence, spread, and control[J]. Journal of Basic Microbiology, 2021, 61(12): 1049-1070. DOI PMID
[15]	张富丽, 尹全, 毛建霏, 等. 转Bt基因棉秸秆还田利用对土壤肥力的影响[J]. 中国生态农业学报(中英文), 2020, 28(5): 734-744.
	ZHANG F L, YIN Q, MAO J F, et al. Effects of Bacillus thuringiensis transgenic cotton straw returning to field on soil fertility[J]. Chinese Journal of Eco-Agriculture, 2020, 28(5): 734-744. (in Chinese with English abstract)
[16]	张凡, 魏萌涵, 孟亚丽, 等. 秸秆还田与施钾对棉花蕾铃分布及根系活力的影响[J]. 农业与技术, 2022, 42(16): 1-5.
	ZHANG F, WEI M H, MENG Y L, et al. Effects of straw returning and potassium application on bud and boll distribution and root activity of cotton[J]. Agriculture and Technology, 2022, 42(16): 1-5. (in Chinese)
[17]	CARADUS J R. Intended and unintended consequences of genetically modified crops-myth, fact and/or manageable outcomes?[J]. New Zealand Journal of Agricultural Research, 2023, 66(6): 519-619.
[18]	GURĂU C, RANCHHOD A. The futures of genetically-modified foods: global threat or panacea?[J]. Futures, 2016, 83: 24-36.
[19]	李朝阳, 李良玉, 刁静静. pH和温度对蛋白结构和功能特性影响的研究进展[J]. 科学技术创新, 2019, 18: 59-60.
	LI C Y, LI L Y, DIAO J J. Research progress on the effects of pH and temperature on protein structure and functional characteristics[J]. Scientific and Technological Innovation, 2019, 18: 59-60. (in Chinese with English abstract)
[20]	严谨, 陈小凤, 熊金萍, 等. 不同灭活温度对新型冠状病毒核酸检测的影响[J]. 检验医学与临床, 2021, 18(2): 214-216.
	YAN J, CHEN X F, XIONG J P, et al. Effect of the thermal inactivation on SARS-CoV-2 at different temperatures on real-time quantitative PCR detection[J]. Laboratory Medicine and Clinic, 2021, 18(2): 214-216. (in Chinese with English abstract)
[21]	YAN L F, IWASAKI H. Thermal denaturation of plasmid DNA observed by atomic force microscopy[J]. Japanese Journal of Applied Physics, 2002, 41(Part 1, No. 12):7556-7559.
[22]	JOSEPH S, KAMMANN C I, SHEPHERD J G, et al. Microstructural and associated chemical changes during the composting of a high temperature biochar: mechanisms for nitrate, phosphate and other nutrient retention and release[J]. Science of The Total Environment, 2018, 618: 1210-1223.
[23]	ZHANG M, SHI A, AJMAL M, et al. Comprehensive review on agricultural waste utilization and high-temperature fermentation and composting[J]. Biomass Conversion and Biorefinery, 2023, 13: 5445-5468.
[24]	DING Z J, XU C, YAN J Y, et al. The LRR receptor-like kinase ALR1 is a plant aluminum sensor[J]. Cell Research, 2024, 34: 281-294.
[25]	PARK S C, LIM H S, MUN S E, et al. Potent antifungal functions of a living modified organism protein, CP4-EPSPS, against pathogenic fungal cells[J]. Molecules, 2023, 28(11): 4289.
[26]	CARADUS J R. Processes for regulating genetically modified and gene edited plants[J]. GM Crops & Food, 2023, 14(1): 1-41.
[27]	LOMBARDO L, TRENTI M, ZELASCO S. GM crops: resistance development and impact on biodiversity[M]//CHAURASIA A, HAWKSWORTH D L, PESSOA DE MIRANDA M. GMOs. Topics in biodiversity and conservation. Cham: Springer International Publishing, 2020: 35-68.
[28]	GUAN J W, SPENCER J L, MA B L. The fate of the recombinant DNA in corn during composting[J]. Journal of Environmental Science and Health Part B, 2005, 40(3): 463-473.

热处理对转基因秸秆中重组蛋白和重组DNA的降解作用

Effect of heat treatment on the degradation of recombinant protein and recombinant DNA in transgenic straws

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 28

相关文章 2

编辑推荐

Metrics

本文评价

[1]	沈峥嵘, 戴远兴, 郭留明, 汪芷瑶, 张恒木. 中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用[J]. 浙江农业学报, 2024, 36(9): 2042-2050.
[2]	刘舒亚, 何汶璐, 陶雨, 黄双慧, 任永强, 耿毅, 黄小丽, 陈德芳, 欧阳萍. 鲤疱疹病毒3型ORF57基因工程亚单位疫苗制备及免疫保护研究[J]. 浙江农业学报, 2022, 34(10): 2138-2148.