库尔勒香梨自交不亲和SFBB-γ基因F-box区和可变区V3 RNAi表达载体的构建及遗传转化  

Construction and genetic transformation of RNAi plant expression vector in self-incompatibility F-box region and variable region V3 of SFBB-γ gene from Korla fragrant pear

作　　者：钟颖[1] 冯建荣[1] 刘海楠[1] 李文慧[1] 吕文娟[1] 田雯 Zhong Ying;Feng Jianrong;Liu Hainan;Li Wenhui;Lyu Wenjuan;Tian Wen(The Specialty Fruit and Vegetable Cultivation Physiology and Germplasm Resources Use the Key Laboratory of the Regiment/ Shihezi Uuniversity College of Agriculture,Shihezi,Xinjiang 832003,China)

机构地区：[1]石河子大学农学院/特色果蔬栽培生理与种质资源利用兵团重点实验室,新疆石河子832003

出　　处：《石河子大学学报（自然科学版）》2018年第4期467-474,共8页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(31272129)

摘　　要：目的为在分子水平应用RNAi(RNA interference)技术调控梨自交不亲和性状,培育自交亲和梨品种。方法基于本实验室获得的库尔勒香梨(Pyrus bretschneideri Rehd)自交不亲和品种花粉决定子SFBB-γ基因c DNA全长序列,选取SFBB-γ基因F-box区和可变区V3分别设计141 bp和120 bp的片段作为干扰序列,以棉花基因组DNA 242 bp的序列作为间隔片段,利用融合PCR (Fusion PCR)构建SFBB-γ基因发卡结构(intron-containing hairpin RNA,ihpRNA),酶切后与pCAMBIA1304植物表达载体相连,构建SFBB-γ基因RNAi表达载体并转化至农杆菌GV3101中。利用叶盘转化法转化到库尔勒香梨无菌叶片中,用GUS组织化学染色法鉴定侵染叶片。测序结果表明干扰F-box区获得的ihpRNA臂长为141 bp,茎环253 bp;干扰可变区V3获得的ihpRNA臂长为120 bp,茎环253 bp。结果说明SFBB-γ基因F-box区和V3区的ihpRNA结构融合成功。通过双酶切检验及PCR证实,SFBB-γ基因F-box区和V3区的RNAi表达载体p CAMBIA1304-RNAi-SFBB构建成功; PCR验证和测序结果也表明重组质粒已经成功转入农杆菌GV3101中,GUS染色检验获得蓝色,目的基因已整合进入库尔勒香梨叶片中。结论成功构建库尔勒香梨SFBB-γ基因F-box区及可变区V3的RNAi表达载体,为诱导香梨SFBB-γ基因转录后基因沉默及培育自交亲和梨品种提供参考。Objective The purpose of this study was to lay a foundation for self-incompatibility improvement and self-compatibility pear cultivars breeding by plant gene engineering technology. Methods One full-length,pollen-specific SFBB-γ（SFBBx-gamma（GenBank accession number：KU756268））cDNA gene was cloned from Korla Fragrant Pear in the present study. The use of RNAi technique to interfere with sequences can achieve the affinity mutation of pears. Then the opposite,interval and positive fragments,after purification and concentration,were fused by the second round PCR reaction without primers for the ihpRNA （intron-containing hairpin RNA） construction. The ihpRNA contained fragments were digested by the corresponding restricted enzymes （Pst I and Kpn I） and inserted into the plant expression vector pCAMBIA1304. The correct recombinant plasmids,after enzyme digestion （Pst I and Kpn I） and PCR identification,were transformed into Agrobacterium tumefaciens GV3101. Results The RNAi expression vector was introduced into Korla Fragrant Pear seedlings by leaf disk transformation method. Histochemical staining of GUS was used for the identification of transgenic plants. The results of digestion and bacteria liquid PCR proved that the RNAi component of SFBB-γ had been recombined with pCAMBIA1304 35S-MCS-NOS-NPTII. The RNAi expression vectors pCAMBIA1304-RNAi-SFBB-F-box and pCAMBIA1304-RNAi-SFBB-V3 were constructed successfully and transformed into GV3101. The results of histochemical staining of GUS showed that target gene （SFBB-F-box or SFBB-V3） were integrated into Korla Fragrant Pear leaves. Conclusion These results laid the foundation for the genetic transformation of RNAi expression vector of self-incompatibility gene,which has important value for the breeding of self-compatible pear cultivars.

关 键 词：库尔勒香梨 自交不亲和 SFBB-γ基因 F-BOX 区 RNAi 表达载体 

分 类 号：S662[农业科学—果树学]