摘要
为了优化黄瓜遗传转化体系以及研究CsHIR1基因在黄瓜中的功能特性,本试验利用In-fusion技术构建植物表达载体pCAMBIA35S-CsHIR1,经过限制性内切酶SalⅠ和BamHⅠ双酶切检测、质粒PCR检测和测序鉴定,结果表明,表达载体pCAMBIA35S-CsHIR1已构建成功并转入农杆菌GV3101中。以黄瓜抗病种质‘PI197088’和感病种质‘CCMC’的子叶节为外植体,通过优化的农杆菌介导法初步将CsHIR1基因转入黄瓜中,经PCR检测获得25株阳性植株,其中8株阳性植株自交得到种子T1代,T1代PCR检测阳性率为44.7%,实时荧光定量分析表明CsHIR1在T1代阳性株中的相对表达量稍高于对照植株,有1株与对照组的差异较显著,这为进一步研究CsHIR1在黄瓜中的功能奠定了基础。
In order to study the genetic CsHIR1 features and optimize the cucumber genetic transfor- mation system, CsHIR1 was studied to transform into cucumber plants. The plant expression vector pCAMBIA35S-CsHIR1 was constructed by In-fusion technology. After double enzymes Sal I and BamH I digestion detection, plasmid PCR detection and sequence identification, the results showed that expression vector pCAMBIA35S-CsHIR1 has been successfully built and transferred into agrobac- terium GV3101. Using the cotyledon node of cucumber germplasm ＇CCMC＇ of susceptible to downy mildew and high-resistant germplasm＇PI197088＇as explant, CsHIR1 gene has been initially proved to transfer into cucumber through the optimization of agrobacterium-mediated genetic transformation system. The 25 positive plants have been obtained by PCR detection, and 8 of which got T1 seeds. The PCR detection positive rate from the T1 generation was 44. 7%0. We examined the expression of CsHIR1 by real time quantitative PCR in seedlings of T1 generation, one of the six lines was higher than CK indicating the CsHIR1 has been transformed into cucumber.
出处
《西北农业学报》
CAS
CSCD
北大核心
2017年第2期255-261,共7页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家自然科学基金(31171955
31471891)
西北农林科技大学优秀人才科研专项资金(QN2009011)~~
关键词
黄瓜
CsHIR1
表达载体
遗传转化
Cucumber
CsHIR1 gene
Expression vector
Genetic transformation
