【摘要】：內(nèi)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)的存在和較低的有機(jī)硫營養(yǎng)水平,限制了山黧豆在干旱、半干旱地區(qū)的推廣。作為β-ODAP生物合成途徑中的關(guān)鍵酶,b-腈基丙氨酸合成酶(b-cyanoalanine synthase,CASase)同時(shí)參與腈基的解毒和Cys代謝,這表明CASase可能在β-ODAP與硫代謝平衡中具有重要功能。因此,獲得Ls CASase基因并對(duì)其進(jìn)行功能研究對(duì)于理解β-ODAP調(diào)控機(jī)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術(shù)結(jié)合RACE方法獲得了Ls CASase基因的c DNA全長,并對(duì)該基因進(jìn)行了生物信息學(xué)分析、組織特異性表達(dá)分析、RNAi載體構(gòu)建及離體再生體系的構(gòu)建等研究,為進(jìn)一步揭示Ls CASase基因表達(dá)與Cys,b-ODAP積累的關(guān)系以及“低毒、高硫”山黧豆的選育奠定分子基礎(chǔ)。主要結(jié)果如下:1.獲得了1551 bp的Ls CASase基因c DNA全長序列。該基因的CDS序列為1146bp,編碼381個(gè)氨基酸。生物信息學(xué)預(yù)測表明,CASase具有CBS-like蛋白功能結(jié)構(gòu)域,不含跨膜結(jié)構(gòu)并定位于線粒體基質(zhì),屬于Trp-synth-beta II超家族。CASase蛋白N端存在一個(gè)磷酸吡哆醛結(jié)合位點(diǎn)、一個(gè)催化位點(diǎn)和一個(gè)單體相互作用位點(diǎn)。進(jìn)化分析結(jié)果顯示,CASase與擬南芥、大豆CAS蛋白親緣關(guān)系較近,因而以擬南芥CAS蛋白結(jié)構(gòu)為模板,預(yù)測了山黧豆CASase蛋白的三維結(jié)構(gòu)。另外,分析了Ls CASase基因組織特異性表達(dá)。結(jié)果表明Ls CASase基因在第6天根中表達(dá)量最高,其后依次是第4天的幼苗、成熟葉、成熟莖;萌發(fā)2天的種子與干種子表達(dá)量幾近相同。2.針對(duì)Ls CASase基因中部序列擴(kuò)增RNAi正反義片段;通過BP反應(yīng)得到入門克隆載體p ENTR-CASase,經(jīng)由LR反應(yīng)得到表達(dá)克隆載體p SGRNAi-CASase。采用凍融法將表達(dá)載體導(dǎo)入農(nóng)桿菌GV3101中,用于山黧豆的遺傳轉(zhuǎn)化。3.獲得山黧豆高分化率的綠色瘤狀愈傷組織。利用腋芽、葉和莖外植體構(gòu)建了山黧豆離體再生體系。并利用上述RNAi載體轉(zhuǎn)化山黧豆愈傷組織,進(jìn)行了抗性篩選。
[Abstract]:The presence of endogenous toxin 尾 -N-oxalyl-L- 偽, 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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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