木糖选择系统下xylA&BADH双价基因对二色胡枝子的遗传转化  

Transformation of Lespedeza bicolor with Bivalent Gene xylA & BADHunder Xylose Selection System

作　　者：杨晓红[1] 陈晓阳[2] Yang Xiaohong;Chen Xiaoyang(College of Landscape, Beijing University of Agriculture ,Beijing 102206;College of Forest, Southern China Agricultural University, Guangzhou 510642)

机构地区：[1]北京农学院园林学院,北京102206 [2]华南农业大学林学院,广州510642

出　　处：《林业科学》2021年第6期74-84,共11页Scientia Silvae Sinicae

基　　金：北京市教委科技项目(SQKM201810020013);北京市教委创新团队项目(IDHT20180509);“863”国家高技术研究发展计划(2002AA241111)。

摘　　要：【目的】建立安全、无抗生素的二色胡枝子遗传转化体系,通过基因工程技术进一步提高二色胡枝子的抗逆性。【方法】采用PCR法从大肠杆菌DH5α菌株中克隆木糖异构酶基因(xylA);构建含有xylA和甜菜碱醛脱氢酶基因(BADH)的植物表达载体pBI121-xylA-BADH;培养基中用木糖取代一定量的蔗糖建立二色胡枝子的木糖选择系统;以农杆菌介导法对二色胡枝子的子叶节进行遗传转化,通过PCR和Southern检测确定转基因植株;对转化植株及非转化植株进行0.5~2 g·L^(-1)NaCl胁迫,观测植株生长表现,测定1 g·L^(-1)NaCl胁迫下转化植株和非转化植株的甜菜碱醛脱氢酶活性、叶绿素、电导率。提取转化植株及非转化植株组培苗叶片中的可溶性总糖,用高效液相色谱仪分析糖成分。【结果】测序结果表明,克隆的木糖异构酶基因与GenBank公布的大肠杆菌xylA核苷酸序列的同源性达到100%,PCR及酶切检测表明正确构建了pBI121-xylA-BADH植物表达载体。在二色胡枝子遗传转化过程中,应在纯木糖培养基上筛选抗性芽,在抗性芽增殖和生根过程中,可以采用蔗糖5 g·L^(-1)、木糖25 g·L^(-1)的糖类混合剂进行进一步筛选。在农杆菌介导的遗传转化中,外植体预培养1天后,采用LBA4404菌株侵染20~30 min,共培养3天,可以获得相对较多的木糖抗性芽。对获得的部分抗性芽进行PCR及Southern检测,得到2个转基因株系,二色胡枝子遗传转化率低于4.7%。耐盐性观测表明,转基因植株能够在含有2 g·L^(-1)NaCl的培养基上生长并生根,而非转基因植株在相同培养基上则叶子变黄、脱落,且不能生根。无盐胁迫时,转基因和非转基因植株在甜菜碱醛脱氢酶活性、叶绿素含量和电导率方面无显著性差异;盐胁迫后,非转基因植株的甜菜碱醛脱氢酶活性无明显变化,而转基因植株的甜菜碱醛脱氢酶活性大幅度提升,且达到非转基因植株的8~10倍;盐【Objective】To establish a safe and antibiotic-free genetic transformation system of Lespedeza bicolor.Using genetic engineering technology to improve the tolerance to abiotic stress of L.bicolor.【Method】The coding sequence of xylose isomerase gene(xylA)was cloned from Escherichia coli DH5αstrain by PCR amplification.The pBI121-xylA-BADH plant expression vector with xylA&betaine aldehyde dehydrogenase gene(BADH)was built.The xylose selection system of L.bicolor was built by using xylose instead of a certain amount of sucrose in the mediums.The cotyledonary node explants of L.bicolor were transformed by using the Agrobacterium tumefaciens-mediated method.The transgenic plants were confirmed by PCR amplification and Southern blot analysis.The growth performance of transgenic and wild-type plants under 0.5-2 g·L^(-1)NaCl stress was measured.The betaine aldehyde dehydrogenase activity,contents of chlorophyll and conductivity of transgenic and wild-type plants in vitro under 1.0 g·L^(-1)NaCl stress were assayed.The total soluble sugars were extracted separately from the leaves of wild-type plants and transgenic plants in vitro.The sugars were assayed by high performance liquid chromatograph.【Result】After DNA sequence analysis,the homology is up to 100%between the cloned product and the published xylA DNA sequence from E.coli in GenBank.The PCR products and restriction enzyme digestion assays showed the pBI121-xylA-BADH plant expression vector was correctly constructed.In the transformation of L.bicolor,the selective mediums for regeneration of adventitious buds from cotyledonary nodes should be the medium which contained xylose as the sole carbon resource,while the selective mediums for shoot segment multiplying or rooting should be the mediums which contained 5 g·L^(-1)sucrose and 25 g·L^(-1)xylose.In Agrobacterium-mediated genetic transformation,the explants were precultured for 1 day,then infected with LBA4404 strain for 20-30 min,and cultured with Agrobacterium tumefaciens for a total of 3 days,rel

关 键 词：二色胡枝子 遗传转化 甜菜碱醛脱氢酶基因 木糖异构酶基因 木糖选择 

分 类 号：S718.46[农业科学—林学]