【摘要】：RNAi是指轉(zhuǎn)入小片段雙鏈RNA而引發(fā)目的基因表達沉默的現(xiàn)象,該現(xiàn)象可以抑制或減弱甜瓜病毒生理生化相關(guān)的基因表達,而達到抗病效果,本研究利用RNAi技術(shù)培育出轉(zhuǎn)基因甜瓜苗,為了解新疆甜瓜上的抗病毒生理機制、抗病毒甜瓜的育種提供研究基礎(chǔ)。本研究主要采用DNAMAN5.0、CLUSTAL X和MEGA5.O軟件對新疆不同主栽區(qū)發(fā)生的WMV、CMV、ZYMV的cp基因進行序列比對,找出保守序列作為目標干擾片段,構(gòu)建RNAi植物表達載體；利用農(nóng)桿菌介導(dǎo)法將目的片段轉(zhuǎn)入甜瓜,最終得到轉(zhuǎn)基因甜瓜苗。本研究得出以下主要研究成果如下：(1)新疆不同地區(qū)WMV、ZYMA、CMVcp基因的堿基序列的同源性分別達到86.25%、93.37%、83.13%以上,同源相似性較高；通過進化關(guān)系分析表明WMV在南北疆均有分布,可分為2個亞組,第1亞組為南疆分離物,第2亞組為北疆分離物；CMV分為4個亞組,3株分離物與北京、山東、山西和臺灣的進化關(guān)系較近；ZYMV可分為3個亞組,部分新疆分離物與3個國內(nèi)外地區(qū)分離物的進化關(guān)系較近；新疆不同地區(qū)WMV、ZYMV、CMV cp基因均存在堿基突變現(xiàn)象。(2)建立WMV、ZYMV、CMV三種病毒多重RT-PCR反應(yīng)體系(25μ1)：模板cDNA最適體積為1.8μ1；引物總體積為2μ1時,比例WMV：CMV：ZYMV=9:6:5較合適；dNTPs最適濃度為0.4 mmol/L；Taq DNA聚合酶單位活力為0.75 U；Mg2+最適濃度為1.5 mmol/L；最適退火溫度為50℃。(3)建立'XHXC'和‘86-1’兩種甜瓜以子葉為外植體的組織培養(yǎng)與再生體系。5%NaClO的濃度下消毒20 min條件下獲得無菌苗最好；‘86-1’和'XHXC'分別在6-BA濃度為1.2 mg/L和1.0 mg/L時愈傷的長勢最好；在MS10培養(yǎng)基上的不定芽誘導(dǎo)率較高；MS19為‘86-1’的最適芽伸長培養(yǎng)基,MS20為'XHXC'的最適芽伸長培養(yǎng)基；MS24為兩種甜瓜最適生根培養(yǎng)基。(4)經(jīng)過分子檢測最終得到‘86-1’甜瓜轉(zhuǎn)pCAMBIA2300WMV-SA基因苗2株,轉(zhuǎn)pCAMBIA2300CMV-SA基因苗2株,轉(zhuǎn)pCAMBIA2300ZYMV-SA基因苗4株；'XHXC'甜瓜轉(zhuǎn)pCAMBIA2300WMV-SA基因苗2株,pCAMBIA2300CMV-SA基因苗3株,pCAMBIA2300ZYMV-SA基因苗2株。
[Abstract]:RNAi refers to the phenomenon of silencing gene expression induced by small fragment double-stranded RNA, which can inhibit or weaken the expression of genes related to physiology and biochemistry of muskmelon virus, and achieve the effect of disease resistance. In this study, transgenic muskmelon seedlings were cultivated by RNAi technique, which provided the basis for understanding the anti-virus physiological mechanism and breeding of anti-virus melon in Xinjiang. In this study, DNAMAN5.0,CLUSTAL X and MEGA5.O software were used to analyze the sequence alignment of WMV,CMV,ZYMV cp gene in different planting areas in Xinjiang, and the conservative sequence was identified as the target interference fragment to construct RNAi plant expression vector. The target fragment was transferred into muskmelon by Agrobacterium tumefaciens and transgenic melon seedlings were obtained. The main results of this study are as follows: (1) the homology of WMV,ZYMA,CMVcp gene in different regions of Xinjiang is over 86.250.373.13%, the homology is high, and the analysis of evolutionary relationship shows that WMV is distributed in both north and south Xinjiang. It can be divided into two subgroups, the first group is the south Xinjiang isolate, the second group is the northern Xinjiang isolate CMV is divided into 4 subgroups and 3 isolates, and the evolutionary relationship between Shandong, Shanxi and Taiwan can be divided into three subgroups. Some Xinjiang isolates were closely related to the evolution of the three isolates at home and abroad, and the WMV,ZYMV,CMV cp gene had base mutation in different regions of Xinjiang. (2) the multiplex RT-PCR reaction system of three WMV,ZYMV,CMV viruses was established (25 渭 1): the optimal volume of template cDNA was 1.8 渭 1; When the total volume of primer is 2 渭 1, the optimum concentration of WMV:CMV:ZYMV=9:6:5 is 0.4 mmol/L;Taq DNA polymerase unit activity, and the optimum concentration is 1.5 mmol/L;. The optimum annealing temperature was 50 鈩，

本文編號：2225217