利用RNAi抑制B-hordein合成降低大麦籽粒蛋白质含量

Creating Low Grain Protein Content Barley by Suppressing B-hordein Synthesis Through RNA Interference

【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。

【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.