胶孢炭疽菌CgATPase调控的α-淀粉酶基因鉴定及作用机理研究

Colletotrichum gloeosporioides is an important plant pathogenic fungus infecting a wide range of hosts, which causes huge economic losses to the natural rubber production. The present study showed that the mutation of the genes of P-type ATPase protein family could affect the filamentous fungi secreting a-amylases to use starch. In our previous study, the results exhibited that the deletion of the CgATPase encoding Ca2+-ATPase (a disease-related gene belonging to the P-type ATPase family) resulted in the decrease of the a-amylases activity. Ca2+-ATPases play an important role in the transportation of Ca2+ and Ca2+ signaling, and Ca2+ is essential for a-amylases protein family keeping activity and stability. In the present it is not clear which a-amylase is regulated by CgATPase. The questions remain whether the over-expression of the CgATPase gene will increase the activity of a-amylase? What is the role of a-amylase gene on the growth and development of C. gloeosporioides in addition to the hydrolysis of starch? Is it a pathogenic factor of C. gloeosporioides? In this study, isolation and functional study of the a-amylase gene regulated by calcium-translocating P-type ATPases gene will be conducted by qRT-PCR, gene deletion / complementation and yeast two-hybrid technology, etc.. Isolating the a-amylase gene from C. gloeosporioides will enrich the fungal a-amylase gene bank, and analysing the mechanism of CgATPase regulating a-amylase gene will provide an important theory for the diseae contry strategy.

胶孢炭疽菌寄主广泛，是重要的植物病原真菌，给天然橡胶生产造成严重经济损失。现有研究表明P型ATP酶家族基因突变会影响丝状真菌分泌α-淀粉酶水解利用淀粉。申请者研究中发现P型ATP酶家族成员Ca2+-ATP蛋白酶CgATPase基因（致病相关基因）缺失导致胶孢炭疽菌α-淀粉酶活性下降。Ca2+-ATP蛋白酶在Ca2+运输和Ca2+信号传递中具有重要作用，而Ca2+对α-淀粉酶家族保持酶活力和稳定性是必须的。目前尚不清楚该基因调控的α-淀粉酶基因，该基因过表达是否会上调α-淀粉酶活性？α-淀粉酶除水解淀粉外，在胶孢炭疽菌生长和发育中有什么作用？是否为胶孢炭疽菌的致病因子？本研究拟用qRT-PCR、基因删除/互补和酵母双杂交等技术，分离CgATPase调控α-淀粉酶基因，鉴定α-淀粉酶功能及分析CgATPase对α-淀粉酶的作用机制，本研究对丰富真菌α-淀粉酶基因库和炭疽病防治具有重要理论意义。

α-淀粉酶和P型ATP蛋白酶家族是生物体中两类重要蛋白酶，α-淀粉酶是淀粉分解代谢中的关键酶，培育α-淀粉酶高产菌株及其表达调控一直是微生物发酵工业中倍受关注的研究热点。P型ATP酶家族成员Ca2+-ATP蛋白酶在Ca2+运输和Ca2+信号传递中具有重要作用。现有研究表明P型ATP蛋白酶基因缺失会影响淀粉的利用。通过基因敲除技术获得了Ca2+-ATP酶MgAPT2基因缺失菌，与野生型Guy11菌株的相比MgAPT2基因缺失菌T-45和T-67在淀粉为唯一碳源的查氏培养基上生长显著受抑制。从Guy11菌株中共找到5个α-淀粉酶基因，MgAmya基因有1579 bp，含有2个内含子。MgAmyc基因有1672 bp，含有1个内含子。MgAmyd基因有2860 bp，含有6个内含子。MgAmye基因有1816 bp，含有2个内含子。MgAmyf基因有2425 bp，含有4个内含子；系统发育分析，MgAmya、MgAmyc和MgAmye聚类到群II；MgAmyd和MgAmyf聚类到群III。实时荧光定量PCR检测结果表明，Ca2+-ATP酶MgAPT2基因敲出后，在1 h，3 h，6 h，12 h，24 h和48 h培养时间点均未能检测到α-淀粉酶MgAmya和MgAmyd基因的表达，α-淀粉酶MgAmyc基因恰恰相反，其表达随MgAPT2基因敲出后上调表达，MgAmye基因在野生Guy11菌株和T-45突变菌中表达量差异不大，MgAmyf基因在野生Guy11菌株和T-45突变菌均未检测到表达。菌株分解淀粉是由α-淀粉酶家族基因协同完成，单一α-淀粉酶基因缺失不足影响其对淀粉的利用、菌株的生长发育及致病力。MgAmya基因在毕赤酵母中不表达，在大肠杆菌中成功表达但形成包涵体。MgAmya和MgAPT2间不存在蛋白互作，Ca2+-ATP蛋白酶很可能是通过阻断钙离子通道和调节细胞内钙离子浓度进而调控α-淀粉酶蛋白基因家族的功能。