山黧豆β-腈基丙氨酸合成酶基因cDNA全长的克隆及功能的初步研究

发布时间：2018-10-10 06:09

【摘要】：内源毒素β-N-草酰-L-α,β-二氨基丙酸(α-N-oxalyl-L-α,β-diaminopropionic acid,β-ODAP)的存在和较低的有机硫营养水平,限制了山黧豆在干旱、半干旱地区的推广。作为β-ODAP生物合成途径中的关键酶,b-腈基丙氨酸合成酶(b-cyanoalanine synthase,CASase)同时参与腈基的解毒和Cys代谢,这表明CASase可能在β-ODAP与硫代谢平衡中具有重要功能。因此,获得Ls CASase基因并对其进行功能研究对于理解β-ODAP调控机理及“低毒、高硫”山黧豆品系的培育十分重要。本研究采用同源克隆技术结合RACE方法获得了Ls CASase基因的c DNA全长,并对该基因进行了生物信息学分析、组织特异性表达分析、RNAi载体构建及离体再生体系的构建等研究,为进一步揭示Ls CASase基因表达与Cys,b-ODAP积累的关系以及“低毒、高硫”山黧豆的选育奠定分子基础。主要结果如下:1.获得了1551 bp的Ls CASase基因c DNA全长序列。该基因的CDS序列为1146bp,编码381个氨基酸。生物信息学预测表明,CASase具有CBS-like蛋白功能结构域,不含跨膜结构并定位于线粒体基质,属于Trp-synth-beta II超家族。CASase蛋白N端存在一个磷酸吡哆醛结合位点、一个催化位点和一个单体相互作用位点。进化分析结果显示,CASase与拟南芥、大豆CAS蛋白亲缘关系较近,因而以拟南芥CAS蛋白结构为模板,预测了山黧豆CASase蛋白的三维结构。另外,分析了Ls CASase基因组织特异性表达。结果表明Ls CASase基因在第6天根中表达量最高,其后依次是第4天的幼苗、成熟叶、成熟茎;萌发2天的种子与干种子表达量几近相同。2.针对Ls CASase基因中部序列扩增RNAi正反义片段;通过BP反应得到入门克隆载体p ENTR-CASase,经由LR反应得到表达克隆载体p SGRNAi-CASase。采用冻融法将表达载体导入农杆菌GV3101中,用于山黧豆的遗传转化。3.获得山黧豆高分化率的绿色瘤状愈伤组织。利用腋芽、叶和茎外植体构建了山黧豆离体再生体系。并利用上述RNAi载体转化山黧豆愈伤组织,进行了抗性筛选。
[Abstract]:The presence of endogenous toxin 尾 -N-oxalyl-L- 伪, 尾 -diaminopropionic acid, 尾 -ODAP and the low level of organic sulfur nutrition limit the popularization of Lathyrus sativus in arid and semi-arid areas. As a key enzyme in 尾 -ODAP biosynthesis pathway, b-cyanoalanine synthase,CASase is involved in the detoxification of nitrile group and the metabolism of Cys, which suggests that CASase may play an important role in the equilibrium of 尾 -ODAP and sulfur metabolism. Therefore, it is very important to obtain Ls CASase gene and study its function in order to understand the regulation mechanism of 尾 -ODAP and the breeding of Lathyrus sativus strain "low toxicity, high sulfur". In this study, the full length of c DNA of Ls CASase gene was obtained by using homologous cloning technique and RACE method. The gene was analyzed by bioinformatics, tissue specific expression analysis, construction of RNAi vector and construction of in vitro regeneration system. In order to further reveal the relationship between Ls CASase gene expression and Cys,b-ODAP accumulation and the breeding of Lathyrus sativus L. The main results are as follows: 1. The full length sequence of c DNA of Ls CASase gene of 1551 bp was obtained. The CDS sequence of the gene is 1146 BP, encoding 381 amino acids. Bioinformatics prediction showed that Casiase had a functional domain of CBS-like protein, did not contain transmembrane structure and was located in mitochondrial matrix. It belonged to the Trp-synth-beta II superfamily. There was a pyridoxal phosphate binding site in the N-terminal of Trp-synth-beta II superfamily. A catalytic site and a monomer interaction site. The result of evolution analysis showed that the relationship between CASase protein and Arabidopsis thaliana and soybean CAS protein was close, so the three-dimensional structure of CASase protein of Lathyrus sativus was predicted by using Arabidopsis thaliana CAS protein structure as template. In addition, the tissue specific expression of Ls CASase gene was analyzed. The results showed that the expression of Ls CASase gene was the highest in the root on the 6th day, followed by the seedling, mature leaf and mature stem on the 4th day, and the expression of Ls CASase gene in the seeds on the 2nd day of germination was almost the same as that in the dry seed. The sense and antisense fragment of RNAi was amplified from the middle sequence of Ls CASase gene, and the expression vector p SGRNAi-CASase. was obtained by LR reaction, and the primer clone vector p ENTR-CASase, was obtained by BP reaction. The expression vector was introduced into Agrobacterium tumefaciens GV3101 by freezing and thawing method, which was used for the genetic transformation of Lathyrus sativus L. A green nodular callus with high differentiation rate was obtained in Lathyrus sativus L. The regeneration system of Lathyrus sativus L. in vitro was constructed by axillary bud, leaf and stem explants. The above RNAi vector was used to transform Lathyrus sativus callus and the resistance screening was carried out.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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