草业学报
草業學報
초업학보
PRATACULTURAL SCIENCE
2015年
8期
142-149
,共8页
许沛冬%郑肖兰%赵艳%李秋洁%吴伟怀%贺春萍%习金根%梁艳琼%郑金龙%戚山江%张晓波%易克贤
許沛鼕%鄭肖蘭%趙豔%李鞦潔%吳偉懷%賀春萍%習金根%樑豔瓊%鄭金龍%慼山江%張曉波%易剋賢
허패동%정초란%조염%리추길%오위부%하춘평%습금근%량염경%정금룡%척산강%장효파%역극현
柱花草%炭疽菌%T-DNA%致病力%基因克隆
柱花草%炭疽菌%T-DNA%緻病力%基因剋隆
주화초%탄저균%T-DNA%치병력%기인극륭
Stylosanthes guianensias%Colletotrichum gloeosporioides%T-DNA%pathogenicity%gene clone
通过对实验室已构建的柱花草炭疽菌 T-DNA 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 T-DNA 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 TAIL-PCR 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。TAIL-PCR 扩增得到 T-DNA 插入位点 RB 端序列467 bp，LB 端侧翼序列388 bp，经两侧序列拼接比对，所得序列与 Pac1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。
通過對實驗室已構建的柱花草炭疽菌 T-DNA 突變體庫中各轉化子緻病力的測定，穫得緻病力喪失突變菌株1869。對其進行 PCR 檢測以驗證 T-DNA 在1869基因組中插入情況，併測定觀察其菌落直徑、菌落形態及產孢量等生物學特性，利用 TAIL-PCR 剋隆標記基因側翼序列，採用生物信息學方法比對基因信息。結果錶明，與柱花草炭疽菌野生型菌株 CH008相比，突變菌株1869錶現緻病力喪失，菌落生長速率也顯著低于野生型；然而，在分生孢子形態、產孢能力、孢子萌髮率等方麵與野生型併無明顯差異。TAIL-PCR 擴增得到 T-DNA 插入位點 RB 耑序列467 bp，LB 耑側翼序列388 bp，經兩側序列拼接比對，所得序列與 Pac1同源性達到92％～96％，推測可能由于基因錶達產物調節菌株對 pH 值的敏感性，從而影響瞭其緻病過程。
통과대실험실이구건적주화초탄저균 T-DNA 돌변체고중각전화자치병력적측정，획득치병력상실돌변균주1869。대기진행 PCR 검측이험증 T-DNA 재1869기인조중삽입정황，병측정관찰기균락직경、균락형태급산포량등생물학특성，이용 TAIL-PCR 극륭표기기인측익서렬，채용생물신식학방법비대기인신식。결과표명，여주화초탄저균야생형균주 CH008상비，돌변균주1869표현치병력상실，균락생장속솔야현저저우야생형；연이，재분생포자형태、산포능력、포자맹발솔등방면여야생형병무명현차이。TAIL-PCR 확증득도 T-DNA 삽입위점 RB 단서렬467 bp，LB 단측익서렬388 bp，경량측서렬병접비대，소득서렬여 Pac1동원성체도92％～96％，추측가능유우기인표체산물조절균주대 pH 치적민감성，종이영향료기치병과정。
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 .
