利用RNAi抑制B-hordein合成降低大麦籽粒蛋白质含量

doi:10.3864/j.issn.0578-1752.2014.19.003

摘要/Abstract

摘要： 【目的】通过RNAi策略抑制大麦籽粒B-hordein的表达，获得高氮肥水平下籽粒蛋白质含量降低的转基因大麦新种质，探索大麦品质改良新途径。【方法】采用同源PCR技术克隆大麦B-hordein核心保守序列，通过Gateway技术构建大麦B-hordein的RNAi植物表达载体pBract207-zz-gp4，利用农杆菌介导法转化大麦Golden Promise幼胚，获得转基因植株。通过PCR检测、Southern杂交验证RNAi构件在转基因大麦及其后代基因组的整合情况。采用半定量RT-PCR分析转基因大麦花后不同发育时期B-hordein转录表达情况。基于近红外谷物分析仪测定蛋白质含量，并进行醇溶蛋白SDS-PAGE检测，以筛选蛋白质含量降低的转基因大麦株系。开展氮肥运筹相关试验，检验RNAi效率及高氮肥水平下转基因大麦蛋白质含量变化情况。【结果】获得大麦B-hordein片段2条（Gp4和Gp5），测序结果表明该片段大小为349 bp，聚类分析表明该序列跟已知大麦醇溶蛋白基因同源性最高达92%，与该基因已登录的mRNA序列同源性高于98%，确认所得片段为大麦B醇溶蛋白基因片段。利用Gateway技术将Gp4片段正反向插入载体pBract207，反向重复序列用i18 和 iv2 intron连接，构建了Ubi启动子驱动的大麦B-hordein RNAi植物表达载体 pBract207-zz-gp4。通过农杆菌介导转化大麦Golden Promise幼胚，结合目标基因及筛选标记基因的PCR验证，获得含有RNAi构件及筛选标记基因的转基因大麦株系11个。Southern杂交检测表明RNAi构件已经稳定整合到T₂/T₃转基因大麦基因组。随机选取6个T₃转基因大麦株系，半定量RT-PCR结果表明花后不同发育时期转基因大麦籽粒B-hordein表达水平明显低于非转基因对照，20 d时转基因株系表达量不仅相比同期对照降低28.19%—55.19%，而且在各时期中也最低。利用随机选取的T₁和T₃籽粒进行蛋白质含量测定表明，8个转基因大麦株系的蛋白质含量相比非转基因对照显著降低，SDS-PAGE电泳结果显示与非转基因相比，转基因各株系B-醇溶蛋白比率有不同程度降低，其中3个株系B-醇溶蛋白比率降低明显，平均减幅为49.42%。采用蛋白质含量降低的转基因大麦株系RNAi-20开展氮肥运筹试验，结果表明，高氮肥水平HN₂及HN₃处理，该株系蛋白质含量显著低于非转基因对照；施以等量高氮肥时，伴随氮肥后移（HN₂到HN₃），该株系总蛋白含量相对非转基因对照逐渐降低。SDS-PAGE电泳结果显示B醇溶蛋白含量降低，电泳带型发生变化。【结论】RNAi技术能抑制籽粒B-hordein表达，降低B-醇溶蛋白含量，高氮肥水平下能显著降低大麦蛋白质含量。

关键词: 大麦, B醇溶蛋白基因, 蛋白质含量, RNAi

Abstract:

【Objective】The objective of the study is to develop a transgenic barley germplasm with low protein content under high nitrogen dosage by RNA interference (RNAi) to specially down-regulate the expression of grain B-hordein genes.【Method】The conserved sequence of B-hordein gene (gi|18928) in barley was cloned based on recombinant PCR and confirmed by sequence analysis. The RNAi expression vector pBract207-zz-gp4, aiming at silencing barley grain B-hordein gene, was reconstructed by Gateway technology and transformed into barley cultivar Golden Promise by Agrobacterium-mediated method using immature embryo. Transgenic plants were obtained after resistance selection, differentiation and regeneration, and then the transgenic progeny was screened by PCR, Southern blotting, and semi-quantitative RT-PCR. Protein content were determined by near-infrared spectroscopy analyzer, SDS-PAGE analysis and nitrogen management.【Result】Two B-hordein fragments (Gp4 and Gp5 ) were identified in this study. Sequence analysis revealed that the length of the sequence was 349 bp. Cluster analysis showed that the highest degree of identity (92%) was between Gp4/Gp5 and other known B-hordein. And also the highest degree of identity (98%) was between conserved sequence and the mRNA of B-hordein. The hpRNA silencing fragment was designed on the basis of Gp4 in sense and antisense orientation with the sequence of the i18 and iv2 intron as spacer region between the repeats and driven by Ubi promoter. Twenty-three T₁ transgenic lines were obtained by Agrobacterium-mediated method and eleven transgenic plants of T₁ generation were confirmed by PCR of selectable marker gene and sense and antisense reaction of B-hordein. Southern blot analysis showed that RNAi constructs were successfully integrated into the plant genomes in T₂/T₃ generations. Further, six transgenic barley positive lines of T₃ progeny were selected by random and analyzed by semi-quantitative RT-PCR after flowering. The results showed that the development expression of B-hordein genes of six T₃ transgenic plants was significantly lower than that of non-transgenic plant, especially on the 20 DAF (Day after flowering), which was not only reduced by 28.19%-55.19% compared with the control, but also was the lowest one among the developing periods after flowering. Then protein content analysis of eight lines of T₁/T₃ selected by random demonstrated that the total protein content decreased significantly compared with the control. At the same time, SDS-PAGE revealed that the ration of B-hordein of different transgenic barley plants or lines decreased compared with that of non-transgenic plants, that of three lines of them reduced significantly and the degree of decreasing was 49.42% on average. In addition, RNAi-20, which had low protein content, was used to carry out nitrogen management analysis. The result revealed that the protein content of the line significantly decreased compared with that of control in high nitrogen dosage (HN₂ and HN₃) treatment, and which gradually lower than the control with the change of nitrogen application from HN₂ to HN₃. SDS-PAGE demonstrated that the B-hordein content of transgenic barleys decreased and electrophoretic band changed.【Conclusion】The RNAi technology can effectively inhibit the expression of B-hordein gene in barley grain, decrease the B-hordein content of RNAi lines, reduce the protein content under high nitrogen dosage, improve the quality of barleys, and create new superordinary germplasm resources of barley.

Key words: barley, B-hordein, protein content, RNA interference

李静雯，张正英，令利军，李淑洁. 利用RNAi抑制B-hordein合成降低大麦籽粒蛋白质含量[J]. 中国农业科学, 2014, 47(19): 3746-3756.

LI Jing-wen, ZHANG Zheng-ying, LING Li-jun, LI Shu-jie. Creating Low Grain Protein Content Barley by Suppressing B-hordein Synthesis Through RNA Interference[J]. Scientia Agricultura Sinica, 2014, 47(19): 3746-3756.

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参考文献

[1] Smith D B. Barley seeds protein and its effects on malting and brewing quality. Plant Varieties and Seeds, 1990, 3(2): 63-80.
[2] Bertholdsson N O. Characterization of malting barley cultivars with more or less stable grain protein under varying environmental conditions. European Journal of Agronomy, 1990, 10: 1-8.
[3] Arends A M, Fox G P, Henry R J, Marschke R J, Synmons M H. Genetic and enviomenttal varlarion in the Dsiastatic power of Australian barley. Journal of Cereal Science, 1995, 21: 63-70.
[4] Vanden Berg R, Muts G C J, Drost B W, Graveland A. Protein from barley to wort//Proceedings of the European Brewery Convention Congress. Copenhagen, IRL Press: Oxford, 1981: 461-469.
[5] 王莉. 关于国产啤酒大麦与进口啤酒大麦品质比较的研究//中国大麦文集: 第五集. 北京: 中国农业科技出版社, 2001: 229-235.
Wang L. Research on the domestic beer barley and the quality of imported beer barley comparison// Barley Science in China: Fifth Set. Beijing: Chinese Agricultural Science Press, 2001: 229-235. (in Chinese )
[6] 蔡剑, 姜东, 戴廷波, 曹卫星. 施氮水平对啤酒大麦植株氮素吸收与利用及籽粒蛋白质积累和产量的影响. 作物学报, 2009, 35(11): 2116-2121.
Cai J, Jiang D, Dai T B, Cao W X. Effects of nitrogen application rates on nitrogen uptake and use, protein accumulation, and grain yield in malting barley. Acta Agronomica Sinica, 2009, 35(11): 2116-2121. (in Chinese)
[7] Masclaux-Daubresse C, Reisdorf-Cren M, Orsel M. Leaf nitrogen remobilization for plant development and grain filling. Plant Biology, 2008, 9: 23-36.
[8] Shewry P R, Faulks A J, Pickering R A, Jone I T, Finch R A, Miflin B. The genetic analysis of barley storage proteins. Heredity, 1980, 44(3): 383-389.
[9] Shewry P R, Kreis M, Parmar S, Lew E J L, Kasarda D D. Identification of γ-type hordeins in barley. FEBS Letters, 1985, 19(1): 61-64.
[10] Bartels D, Thompson R D. Synthesis of messenger-RNAs coding for abundant endosperm proteins during wheat grain development. Plant Science, 1986, 46: 117-125.
[11] Sørensen M B, Cameron-Mills V, Brandt A. Transcriptional and post-transcriptional regulation of gene expression in developing barley endosperm. Molecular and General Genetics, 1989, 217: 195-201.
[12] Carbonero P, Vicente-Carbajosa J, Mena M, Onate L, Lara P, Diaz I. bZIP and DOF transcription factors in the regulation of gene expression in barley endosperm// Black E M, Bradford K J, Vazquez- Ramos J. Seed Biology: Advances and Applications. Proceeding of the Sixth Interantional Workshop on Seeds. New York: CABI Publishing, 2000: 27-41.
[13] Mehrotra R, Kumar S, Mehrotra S, Singh B D. Seed storage protein gene regulation-A jig-saw puzzle. Indian Journal of Biotechnology, 2009, 8: 147-158.
[14] Segal G, Song R, Messing J. A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics, 2003, 165(1): 387-397.
[15] Gil-Humanes J, Piston F, Hernando A, Avarez J B, Shewry P R, Franciscol B. Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat. Journal of Cereal Science, 2008, 48(3): 565-568.
[16] Wu Y, Messing J. Rescue of a dominant mutant with RNA interference. Genetics, 2010, 186(4): 1493-1496.
[17] Mette L, Vincze E, Wiser Herbert, Jnak S, Preben B H. Suppression of C-Hordein synthesis in barley by antisense constructs results in a more balanced amino acid composition. Journal of Agricultural and Food Chemistry, 2007, 55(15): 6074-6081.
[18] 王关林, 方宏筠. 植物基因工程: 第二版. 北京: 科学出版社, 2002: 776 -777.
Wang G L, Fang H J. Plant Gene Engineering: 2nd ed.. Beijing: Scientific Press, 2002: 776-777. (in Chinese)
[19] 李静雯, 张正英, 李淑洁. 利用RNAi技术沉默大麦B-hordein基因的研究. 麦类作物学报, 2014, 34(2): 169 -174.
Li J W, Zhang Z Y, Li S J. Silence of B-Hordein gene in barley induced by RNAi strategy. Journal of Triticeae Crops, 2014, 34(2): 169-174. (in Chinese)
[20] Bartlett J G, Alves S C, Smedley M, Snape J W, Harwood W A. High-throughput Agrobacterium-mediated barley transformation. Plant Methods, 2008, 4(22): 1-12.
[21] Murray M G, Thompson W F. Rapid isolation of high molecular weight DNA. Nucleic Acids Research, 1980, 8(19): 4321-4325.
[22] Tang G L, Gad G. Using RNAi to improve plant nutritional value: from mechanism to application. Trends in Biotechnology, 2004, 22(9): 24-29.
[23] Jagtap U B, Gurav R G, Bapat V A. Role of RNA interference in plant improvement. Naturwissenschaften, 2011, 98: 473-492.
[24] 孙重霞, 杨凤萍, 张婷, 隋晓燕, 梁荣奇, LIU Qing, 张晓东, 李保云. 利用 RNAi 技术抑制籽粒PPO合成改良小麦面粉白度的研究. 中国农业科学, 2013, 46(6): 1104 -1113.
Sun C X, Yang F P, Zhang T, Sui X Y, Liang R Q, Liu Q, Zhang X D, Li B Y. Down-regulation of the expression of grain ppo genes to improve wheat dough whiteness by RNA interference. Scientia Agricultura Sinica, 2013, 46(6): 1104-1113. (in Chinese)
[25] Winkler H, Powell S. Applications of RNA interference. Targets, 2003, 2: 42-44.
[26] Kreis M, Shewry P R, Forde B G, Rahman S, Bahramian M B, Miflin B J. Molecular analysis of the effects of the lys3A gene on the expression of Hor loci in developing endosperms of barley (Hordeum Vulgare L.). Biochemical Genetics, 1984, 22: 231-255.
[27] Brandt A. Endosperm protein formation during kernel development of wild type and a high-lysine barley mutant. Cereal Chemistry, 1976, 53(6): 890-901.
[28] Kreis M, Shewry P R, Forde B G, Rahman S, Miflin B J. Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (Hordeum Vulgare L.). Cell, 1983, 34(1): 161-167.
[29] Faulks A J, Shewry P R, Miflin B J. The polymorphism and structural homology of storage polypeptides (hordein) coded by the Hor-2 locus in barley (Hordeum vulgare L.). Biochemical Genetics, 1981, 19: 841-858.
[30] Forde B G, Heyworth A, Pywell J, Kreis M. Nucleotide sequence of a B1-hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucleic Acids Research, 1985, 13: 7327-7339.