农杆菌介导的GbWOX9基因对海岛棉胚性细胞的遗传转化

Genetic Transformation of Agrobacterium tumefaciens Mediated GbWOX9 Gene to Embryogenic Cells in Sea Island Cotton

为探究WOX9基因在棉花体细胞胚胎发育过程中的功能,借助农杆菌介导化学试剂诱导型植物表达载体p TA7002/GbWOX9和pTA7002/GbWOX9 anti对海岛棉‘新海16’胚性细胞进行遗传转化,以此利用化学诱导剂在胚性细胞中诱导GbWOX9的过量或抑制表达。研究结果表明在菌液OD600≈0.5~0.6时进行15 min的农杆菌侵染并共培养24 h之后,将侵染的细胞在500 mg/L头孢和20 mg/L潮霉素的抗性培养基上筛选继代两次,最终获得若干抗性阳性胚性细胞。进一步将抗性阳性细胞在化学诱导剂30μmol/L地塞米松处理5 h后,通过qRT-PCR技术对其进行GbWOX9的基因表达鉴定,结果表明与对照组相比,正、反义转基因抗性阳性细胞中的GbWOX9基因表达量皆有显著差异。该研究初步建立了以潮霉素为筛选标记的海岛棉遗传转化体系,并在此基础上将GbWOX9正义和反义链成功导入‘新海16’胚性细胞中,为其功能的鉴定提供了参考。

In order to investigate the function of WOX9 gene in cotton somatic embryogenesis, the study carried out genetic transformation of‘Xinhai 16＇embryogenic cells of sea island cotton by using agrobacterium to mediate chemical reagent induced plant expression vector of pTA7002/GbWOX9 and p TA7002/GbWOX9 anti-vector so as to induce the excessive or inhibitory expression of GbWOX9 in embryo cells by chemical inducer. The results showed that the agrobacterium infection was carried out in OD600≈0.5~0.6 bacterium solution for 15 min. After 24 h co-culture, the infected cells were screened for twice subculture on the resistant medium with 500 mg/L cephalosporin and 20 mg/L hygromycin. Eventually the study obtained several resistance positive embryogenic cells. After treating the resistance positive embryogenic cells in the chemical inducer of 30 μmol/L dexamethasone for 5 hours, the study identified the GbWOX9 gene expression of the resistant positive cells by q RT-PCR technology.And the results showed that the expression of GbWOX9 gene had significant differences both in the sense and antisense transgenic resistant cells compared with the control group. This study preliminarily established the genetic transformation system of sea island cotton with hygromycin as the selection marker, and successfully transferredGbWOX9 sense and antisense strand into the ‘Xinhai 16＇embryogenic cells, which provided reference for the function identification.