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

doi:10.13880/j.cnki.65- 1174/n.2018.04.011

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

钟颖, 冯建荣 , 刘海楠, 李文慧, 吕文娟, 田雯

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石河子大学学报 ›› 2018, Vol. 36 ›› Issue (4) : 467-474. DOI: 10.13880/j.cnki.65- 1174/n.2018.04.011

农业·林业

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

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Constr uction and genetic transformation of RNAi plant expression vector in self- incompatibility F- box region and variable region V3 of SFBB- γ gene fr om Korla fragrant pear

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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 cDNA 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 pCAMBIA1304- 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。

Abstract

O bjective 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.

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钟颖, 冯建荣 , 刘海楠, 李文慧, 吕文娟, 田雯. 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. 石河子大学学报. 2018, 36(4): 467-474 https://doi.org/10.13880/j.cnki.65- 1174/n.2018.04.011

Zhong Ying, Feng Jianrong, Liu Hainan, Li Wenhui, Lyu Wenjuan, Tian Wen. Constr uction and genetic transformation of RNAi plant expression vector in self- incompatibility F- box region and variable region V3 of SFBB- γ gene fr om Korla fragrant pear. Journal of Shihezi University. 2018, 36(4): 467-474 https://doi.org/10.13880/j.cnki.65- 1174/n.2018.04.011

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国家自然科学基金项目（31272129

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Received	Published
2018-03-22	2018-08-01
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2023-12-10

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