番茄MPK2基因煙草遺傳轉(zhuǎn)化體系的建立

1、 番茄MPK2基因煙草遺傳轉(zhuǎn)化體系的建立 摘要：為建立高效快速的番茄MPK2基因煙草遺傳轉(zhuǎn)化體系。首先珊西煙草無菌苗的培養(yǎng)與再生，制備根癌農(nóng)桿菌感受態(tài)，將質(zhì)粒pCAMBIA2301-MPK2導(dǎo)入到根癌農(nóng)桿菌中。然后通過根癌農(nóng)桿菌介導(dǎo)法，卡那霉素的篩選以及多代培養(yǎng)得到MPK2的轉(zhuǎn)基因煙草。最后提取轉(zhuǎn)化煙草葉片DNA，進(jìn)行PCR擴增并鑒定。本試驗總結(jié)了已有的經(jīng)驗建立高效快速的番茄MPK2基因煙草遺傳轉(zhuǎn)化體系，對進(jìn)一步探索番茄MPK基因功能與作用及其應(yīng)用作出一定的貢獻(xiàn)。10972關(guān)鍵詞：MPK2基因；煙草；PCR；轉(zhuǎn)化Establishment of Tomatoes MPK2 Gene Tobac

2、co Genetic Transformation SystemAbstract：The article was to establish a efficient and rapid tobacco genetic transformation system of tomato MPK2 gene. First, Nicotiana tabacum cv. Xanthi-ncsterile plants were cultivated and regenerated. After preparing Agrobacterium tumefaciens competent cells, we t

3、ransfered the plasmid pCAMBIA2301-MPK2 into the Agrobacterium tumefaciens. Then, though Agrobacterium tumefaciens-mediated , kanamycin and generational cultured, we got transformational tobacco. Finally, we extracted, amplified and identified the DNA of the transformational leaf by polymerase chain

4、reaction (PCR). The research summarized the existing experience to establish efficient rapid tobacco genetic transformation system of tomatoes MPK2 gene, to further explore the tomato MPK genes function and to make certain contribution in the application.Key words: MPK2 gene; Tobacco; Polymerase cha

5、in reaction; Transformation目錄摘要1Abstract1引言11材料與方法31.1試驗材料與儀器31.2 試驗方法31.3轉(zhuǎn)基因植株的獲得及篩選42 結(jié)果與分析5 本試驗以煙草為試驗材料，用GV3101根癌農(nóng)桿菌介導(dǎo)重組載體的轉(zhuǎn)導(dǎo)，希望能為植物MAPK C族基因的研究作出一定的貢獻(xiàn)。近年來，國內(nèi)外的學(xué)者對煙草轉(zhuǎn)化體系的研究已經(jīng)較為成熟，本試驗借鑒已經(jīng)研究出的結(jié)果建立的番茄MPK2基因煙草遺傳轉(zhuǎn)化體系期望能對番茄MPK2基因的研究可以對植物MAPK基因功能作進(jìn)一步詮釋奠定一定的基礎(chǔ)。1材料與方法1.1試驗材料與儀器1.1.1材料煙草品種珊西、質(zhì)粒pCAMBIA2301-

6、MPK2、轉(zhuǎn)化受體菌大腸桿菌、GV3101根癌農(nóng)桿菌等試驗材料，均由周口師范學(xué)院植物遺傳與分子育種重點實驗室提供。1.1.2主要儀器瓊脂糖凝膠電泳儀；中速離心機；搖床；無菌工作臺；PCR儀；分光光度計；pH計。1.2 試驗方法1.2.1無菌苗的培養(yǎng)將煙草種子用體積分?jǐn)?shù)75%酒精浸泡30s，用蒸餾水沖洗23遍，再用5%次氯酸鈉溶液消毒810min，用無菌水沖洗35次，將種子散播在MS培養(yǎng)基上。待其長出小苗，轉(zhuǎn)移至盛有MS培養(yǎng)基(pH=5.8)的花瓶中，放于25，周期為16h的光照，8h的黑暗的組培間中，得到煙草無菌苗，待用。1.2.2珊西煙草的再生配制MS+6BA(3 mg/L)+蔗糖3%+NAA(0.2 mg/L)培養(yǎng)基，調(diào)培養(yǎng)基pH=6.06。將無菌苗于無菌水中切成1.0cm×1.0cm左右的葉盤直接放于配制好的培養(yǎng)基中，25，周期為16h的光照，8h的黑暗的組培間中培養(yǎng)觀察再生情況。1.2.3根癌農(nóng)桿菌活化隨機挑取含有質(zhì)粒pCAMBIA2301-MPK2的GV3101根癌農(nóng)桿菌單菌落接種于LB添加有2