扁桃AcCBF1基因VIGS载体构建与功能分析  

Vector construction and functional analysis of AcCBF1 using VIGS method in almond flower organs

作　　者：宋恬 田嘉[1] 李鹏[1] 刘梦婕 张琦[1] 郭长奎[2] 李疆[1] SONG Tian;TIAN Jia;LI Peng;LIU Mengjie;ZHANG Qi;GUO Changkui;LI Jiang(Xinjiang Agricultural University,Urumqi 830052,Xinjiang,China;School of Agriculture and Food Science,Zhejiang Agriculture and Forestry University,Hangzhou 311300,Zhejiang,China)

机构地区：[1]新疆农业大学,乌鲁木齐830052 [2]浙江农林大学农业与食品科学学院,杭州311300

出　　处：《果树学报》2019年第4期421-429,共9页Journal of Fruit Science

基　　金：国家自然科学基金(31660562);新疆自治区园艺学重点学科基金(2016-10758-3)

摘　　要：【目的】利用VIGS(TRV-mediated Virus Induced Gene Silencing)方法沉默扁桃(Amygdalus communis L.)AcCBF1基因表达,并初步探讨AcCBF1对低温条件下花药发育的调控。【方法】从‘纸皮’扁桃花药中克隆AcCBF1基因(729 bp),并设计4个不同长度(729、345、464、270 bp)的AcCBF1基因片段,连接到pTRV2载体上,构建VIGS载体,转化根瘤农杆菌GV3101,并用注射法侵染到扁桃花蕾内。对侵染的花器官低温处理,用半定量PCR和荧光定量PCR分析AcCBF1基因表达,以及对花器官表型进行初步分析。【结果】成功构建了3个AcCBF1基因沉默表达载体,pTRV2-AcCBF12和pTRV2-AcCBF14能显著沉默扁桃花药中AcCBF1的表达;低温处理后这两组处理的扁桃花瓣小且颜色浅,花芽和花药鲜重轻且小,与对照相比差异显著。【结论】AcCBF1在调控低温条件下对花器官表型指标发挥重要作用。该研究结果可为扁桃生殖期抗冻相关基因的功能验证和分子调控机制研究提供重要的参考和技术支持。【Objective】To clone some fragments of the almond AcCBF1 gene and construct the gene silencing vector pTRV2-AcCBF1 mediated by tobacco rattle virus , preliminary study was carried out on the AcCBF1 regulation of flower organ development under low temperature conditions, so as to lay a theoretical foundation for the breeding of hardy- resistant varieties and further study on gene function verification【. Methods】The target gene fragment was cloned from the anther of‘Zhipi’almond by RTPCR. After the constructed expression vector was transfected into Agrobacterium GV3101, the anthers were infected by means of injection and vacuum infection. The empty vector control and infested shoots were placed in an artificial climate chamber with 70% relative humidity for hydroponics until the budswell stage(16 h during the day, 23 ℃, 8 000 lx;8 h at night, 15 ℃, 0 lx). After the end of the night mode, the artificial climate box was adjusted to 10 ℃, 0 lx, 12 h. Then, the anthers of 30 branches of each treatment group were stripped and stored with liquid nitrogen. The remaining branches were transfered to the gradient refrigerator at 4 ℃· h-1 to 0 ℃,0 ℃ 3 h and -2 ℃ 2 h. The remaining shoots were warmed at 4 ℃· h- 1, and the culture was continued to observe the phenotypic changes of the branches. Semi-quantitative and fluorescence quantitative analysis were performed with RNA using RT-PCR and q RT-PCR, using PdActin1 as an internal reference gene. The silencing effect of the VIGS system on the AcCBF1 gene was evaluated by phenotypic observation, semi-quantitative and fluorescence quantitative detection.【Results】The results showed that:(1) The expression level of AcCBF1 after low temperature treatment was significantly higher than that before treatment, and there was a significant difference between each treatment and the no- load control after low temperature treatment. The expression of Ac- CBF1 gene in almond anther was analyzed by real-time PCR. The results showed that the expression of AcCBF1

关 键 词：扁桃 AcCBF1 VIGS 低温 花器官 

分 类 号：S662.9[农业科学—果树学]