柱花草炭疽菌致病力丧失突变菌株1869的T-DNA插入位点侧翼序列的克隆  

Molecular characterization of the flanking gene of T-DNA insertional,pathogenicity-defective Colletotrichum gloeosporioides mutant strain 1869

作　　者：许沛冬 郑肖兰[2] 赵艳[1] 李秋洁[2] 吴伟怀[2] 贺春萍[2] 习金根[2] 梁艳琼[2] 郑金龙[2] 戚山江 张晓波[3,4] 易克贤[2,5] 

机构地区：[1]海南大学农学院,海南海口570228 [2]中国热带农业科学院环境与植物保护研究所,海南海口571101 [3]热带作物种质资源保护与开发利用教育部重点实验室(海南大学),海南海口570228 [4]海南大学旅游学院,海南海口570228 [5]中国热带农业科学院热带生物技术研究所,海南海口571101

出　　处：《草业学报》2015年第8期142-149,共8页Acta Prataculturae Sinica

基　　金：国家自然科学基金(31072076;31101408);公益性行业科研专项(201303057);中央级公益性科研院所基本科研业务专项(2015hzs1J002)资助

摘　　要：通过对实验室已构建的柱花草炭疽菌TDNA突变体库中各转化子致病力的测定，获得致病力丧失突变菌株1869。对其进行PCR检测以验证TDNA在1869基因组中插入情况，并测定观察其菌落直径、菌落形态及产孢量等生物学特性，利用TAILPCR克隆标记基因侧翼序列，采用生物信息学方法比对基因信息。结果表明，与柱花草炭疽菌野生型菌株CH008相比，突变菌株1869表现致病力丧失，菌落生长速率也显著低于野生型；然而，在分生孢子形态、产孢能力、孢子萌发率等方面与野生型并无明显差异。TAILPCR扩增得到TDNA插入位点RB端序列467bp，LB端侧翼序列388bp，经两侧序列拼接比对，昕得序列与Pacl同源性达到92％～96％，推测可能由于基因表达产物调节菌株对pH值的敏感性。从而影响了其致病过程。Colletotrichum gloeosporioides is a fungal pathogen of Stylosanthes guianensis causing anthracnose disease.Pathogenicity defective mutant strains of C.gloeosporioides were screened in the laboratory and strain 1869 selected for study.The mutant strain was compared with wild type strain CH008 for phenotypic charac-teristics,including colony diameter,colony morphology,sporulation ability and spore germination rate.Colo- <br> ny diameter of CH008 and strain 1869 differed,but the other traits did not.DNA of the mutant strain was ex-tracted,and using thermal asymmetric interlaced PCR (TAIL-PCR)a T-DNA insertion mutation site be-lieved responsible for loss of pathogenicity was identified.The RB and LB flanking sequences at the insertion site were cloned,also using TAIL-PCR.The RB flanking sequence was 467 bp in length,while the LB flan-king sequence was 388 bp.The BLAST result showed that the sequence had 92% -96% homology to gene Pac1.By predicting the function of a protein coded by the flanking sequence,we inferred that the gene in ques-tion regulated the sensitivity to pH,and in this way affected the pathogenic potential of C.gloeosporioides .

关 键 词：柱花草 炭疽菌 T-DNA 致病力 基因克隆 

分 类 号：S435.4[农业科学—农业昆虫与害虫防治]