摘要
大豆是主要的油料作物之一,而土壤盐渍化和线虫病严重抑制了大豆的生长和发育,因此,培育耐盐抗线虫大豆新品种对提高大豆产量无疑将产生重要影响。本文的目的是利用农杆菌介导的大豆胚尖高效遗传转化方法,将分别来自三种耐盐性不同的植物的与阴离子清除有关的甲基转移酶基因导入大豆中,以期为植物基因工程探索出一条新途径,同时为大豆的遗传育种提供新资料。
对大豆的灭菌方法、外植体的优化、分化和拔高培养基的优化、生根培养基及生根方法等进行了探索,以建立大豆胚尖高效再生体系;对大豆遗传转化过程中的重要因素如筛选压、菌液浓度、浸染时间、农杆菌抑制剂的使用等进行了研究;分别将来自拟南芥、盐芥和盐地碱蓬的甲基转移酶基因MCT利用农杆菌介导的方法导入大豆中,并利用PCR、RT-PCR、Southern杂交等分子手段对转化子进行了检测。
建立了大豆胚尖高效再生体系:成熟大豆种子浸泡24h后,用1:1的1.2%的次氯酸钠和0.1%的升汞混合液处理大豆10min,收集胚尖并转入芽诱导分化培养基MS+0.08 mg/L TDZ,30d后将长有丛生芽的胚尖转入拔高培养基MS+2.0 mg/L KT +0.2 mg/L NAA中,20d后切取苗高大于5cm的植株,转入生根培养基1/2MS+0.5mg/LNAA中,30d后待根长到3-4cm后移栽到培养土中;优化了大豆胚尖遗传转化体系:卡那霉素选择浓度为90mg/L,菌液浓度为OD_(600)=0.6,浸染时间为12h,可用氨苄青霉素作为农杆菌的抑制剂;AtMCT、ThMCT和SsMCT三种不同的基因转化植株经过筛选分别得到78、67、82株抗性苗,PCR、RT-PCR、Southern杂交等分子手段对转化子的检测结果表现为阳性。
首次利用TDZ诱导大豆胚尖的分化,建立了高效的大豆胚尖再生体系,丛生芽分化率高达92.6%;优化了大豆胚尖遗传转化系统,转化率高达15%;首次将来自拟南芥、盐芥和盐地碱蓬的阴离子清除有关的基因甲基转移酶基因在大豆中进行了异源表达,分子检测结果表明外源基因已转入大豆中并进行了表达。
Soybean is an important oil crop plant. High soil salinity and the eelworm limit severely soybean growth, development and productivity. Therefore, developing new cultivars with enhanced salt stress and eelworm tolerance would undoubtedly have an enormous impact on global soybean production. The object pf this study was utilizing the high effective Agrobacterium-mediated transformation system of soybean embryo tip to introduce three methytransferase genes related to anion scavenging from different salt-tolerant plants into soybean,in order to explore a new route for plant gene engineering, and at the same time to provide new information for soybean genetic breeding.
The study has explored the sterilization method, the optimization ofexplant, the optimization of medium and rhizogenesis method, in order to establish an high effective regenesis system of soybean embryo tip;we investigate the important factors such as filtration press, the concentration of Agrobacterium, dip-dying time and the employment of Agrobacterium inhibitor on the course of soybean genetic transformation;we introduce the methytransferase genes related to anion scavenging seperately from Arabidopsis thaliana, Thellungiella salsuginea and Suaeda salsa into soybean by Agrobacterium-mediated transformation system,and the transformation plants were tested by PCR、RT-PCR、Southern blotting analysis.
We have established the high effective regenesis systemof soybean embryotip:the mature soybean seeds were soaped for 24h, then were dealed with 1.2% NaClO and 0.1% HgCl2 (1:1 mixed) for 10min. The embryo tips were collected and cultured on MS medium supplemented with 0.08 mg/L TDZ for 30d, then explants with multiple mud tissues were transferred to MS medium supplemented with 2.0 mg/LKT + 0.2 mg/L NAA for 20d. The induced seedings long than 5cm were transferred onto root-inducing medium (1/2MS+NAA 0.5mg/L) for 30d. Finally,they were transplanted into soil when the roots reached 3-4cm;We also opmized the transformation system of soybean embryo tip:the concentration of kanamycin was 90mg/L,the concentration of Agrobacterium wasOD_(600)=0.6,the dip-dying time was 12h, ampicillin can act as Agrobacterium inhibitor;After screening AtMCT、ThMCT and SsMCT transformation plants were78、67、82seperately,the results of PCR、RT-PCR、Southern blotting analysis on transfoemation plants were positive.
This is the first time that we induce and differentiate soybean embryo tip with TDZ. the study establish high effective regenesis system of soybean embryo tip,the differentiation rate of multiple bud is higher than 92.6%;optimize the Agrobacterium-mediated transformation system of soybean embryo tip,the transformation rate is higher than 15%;the first time to heterologus express the methytransferase genes related to anion scavenging seperately from Arabidopsis thaliana, Thellungiella salsuginea and Suaeda salsa into soybean,The results of molecular analysis indicate that heterologus genes had been integrated into the transgenic plant genomes and expressed.
引文
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