【作者】 王艳；

【导师】 汤继华；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2014， 硕士

【摘要】 玉米作为重要的粮食作物之一，在农业生产和国民经济发展中具有十分重要的作用。用转基因的方法导入优良基因是创造玉米新种质和功能验证的有效手段之一，在玉米遗传学研究和育种中得到了广泛应用。细胞质雄性不育是作物杂种优势利用的一种主要途径，玉米C型胞质雄性不育因其育性稳定成为目前生产上利用的一种主要胞质类型。课题组前期利用图位克隆的方法对玉米C型胞质雄性不育的恢复基因Rf4进行了克隆，本试验拟以玉米幼胚胚性愈伤再生体系为基础，用农杆菌介导法进行Rf4基因的遗传转化，并获得抗性植株，进行基因功能验证，并建立一套稳定、高效的转基因技术体系。试验以多种基因型幼胚为材料，进行胚性愈伤组织的诱导及再生，对转化受体系统的建立进行了探讨，研究了影响转化过程的多个因素，并用农杆菌介导法转化了Rf4基因。主要研究结果如下：1、以自交系综3、87-1、HIIA和杂交组合87-1×综3、综3×87-1、87-1×HIIA基因型的材料进行了受体转化系统的探讨。结果表明：不同基因型玉米幼胚，胚性愈伤诱导率差异性很大。其中，杂交组合综3×87-1和87-1×HIIA的愈伤诱导率和胚性愈伤诱导率相对较高，分别达97.5%、65.5%和98.4%、70.9%；而87-1和87-1×综3的胚性愈伤诱导率则较低，最低只有20.2%；综3和HIIA的愈伤诱导能力中等。因此，综3、HIIA、综3×87-1和87-1×HIIA都可以作为良好的受体材料。试验还表明，胚龄和幼胚大小对愈伤诱导率具有一定的影响，幼胚大小在1.0-2.0mm之间最适合诱导愈伤组织，但不同基因型幼胚达到1.0-2.0mm大小的胚龄有差异。在培养基配方方面，2,4-D浓度为1-2mg/L时比较有利于愈伤的诱导，6-BA浓度为1.0mg/L时愈伤的成苗分化率最高，而再生植株生根壮苗最适合的NAA浓度是0.6mg/L。2、以综3×87-1愈伤为受体材料进行的农杆菌介导转化结果表明，菌液浓度OD600为0.6，侵染时间10min，共培养温度为22℃，共培养3天的转化效果最好。试验还确定了该材料的PPT临界筛选浓度为8mg/L，此浓度比较有利于抗性愈伤的筛选。3、利用建立的遗传转化体系，对Rf4基因过表达载体和RNAi载体进行了遗传转化。共获得656株转基因苗，用标记基因Bar和终止子NOS引物进行PCR检测有10株呈阳性，初步认为根癌农杆菌Ti质粒上携带目的基因（Rf4基因）的T-DNA已经被导入。其中RNAi载体转化的6株阳性植株得到了种子。T1和T2代的分子检测和育性调查结果显示，Rf4恢复基因的RANi沉默基因导入阳性植株可导致雄性不育，表明该候选基因可能是恢复基因Rf4。更多还原

【Abstract】 Maize is one of the important food crops, and plays an important role onagricultural production and national economic development. Transgenic approach is aneffective means for modified maize germplasm. The utilization of cytoplasim male sterility(CMS) is the main way for using hybrid, while heterosis is an approach to improve highgrain yield and quality of several crops. In maize, the Rf4restorer gene is one importantgene for Cms-C type. In this study, the transformation technology based onAgrobacterium-mediated was modified, and the candidate gene for Rf4was transformated tomaize by means of Agrobacterium-mediated genetic transformation. The main resultsobtained were as follows:This experiment researched the receptor transformation systems using several genotypesin maize, including inbred lines Zong3,87-1, HIIA, and crosses87-1*Zong3, Zong3*87-1and87-1*HIIA. The results showed that embryo callus induction rate of difference genotypeimmature embryo had significant difference. The callus induction rate and embryonic callusinduction rate of Zong3*87-1and87-1*HIIA were97.5%,65.5%and98.4%,70.9%respectively; but embryonic induction rate of inbred lines87-1and the cross87-1*Zong3were low, the lowest was only20.2%. The callus induction rate of inbred lines Zong3andHIIA was at the middle level. Therefore, the inbred line Zong3, HIIA, and crosses87-1*Zong3as well as87-1*HIIA could be used as good transgenic receptors. Theimmature embryos of1.0-2.0mm in length were optimal for callus induction, when reachingthe length, the immature embryos of different genotypes had different embryo age.1-2mg/lof2,4-D concentration could be conducive to callus induction.1.0mg/L of6-BAconcentration could be benefit to the seedling callus differentiation, and the suitable NAAconcentration for root regenerated was0.6mg/L.This experiment demonstrated that a series of factors could affect the transformationprocess by using the cross Zong3×87-1callus as receptor. The best transformed conditionswere the bacterial concentration OD600of0.6, infection time of10min, co-culturetemperature of22℃, and cultured for3days. The PPT test also identified the criticalscreening concentration of the material was8mg/L, and the concentration was the most conducive resistant callus screening.The callus of cross Zong3×87-1was used as infecting receptor material for transformedthe Agrobacterium EHA105with Rf4candidate gene, and a total of656transgenic seedlingswere obtained. For detection of true transgenic plants, the marker of Bar gene and theprimer of terminator NOS were used, and10transgenic seedlings were identified.6of theidentified planted the seeds. Combination of T1and T2of molecular detection and fertilitysurvey results, drawed Rf4RANi of silence genes into transgenic plants can lead to malesterility, it could be a candidate genes Rf4restoring genes.更多还原