芦笋茎枯病菌聚酮体合成酶基因pks-pa的克隆、表达及功能鉴定

The polyketide biosynthesis (PKS) from the plant pathogenic fungus is mainly included in the biosynthesis of toxins, pigments and 6-methyl salicylates. However, the function of PKS gene pks-pa from asparagus stem blight pathogen has not been known. In the previous study, the type of PKS from asparagus stem blight pathogen has been identified as Type WA and the partial suquence has been obtained through PCR using specific and degenerate primers. On the basis of the previous research, the full cDNA and gDNA sequences will firstly be obtained through 5'/3'-RACE and Tail-PCR techniques, followed by bioinformatics analysis with public gene databases and related software for a comprehensive understanding of the genetic characteristics. Then, the expression vector of pks-pa gene from asparagus stem blight pathogen will be constructed and the positive strains for induced expression will be obtained by screening and verification of engineering bacteria, followed by the study of protein characteristics from PKS through the analysis of protein solubility and western blotting, etc. Finally, the knock-out vector will be constructed and the gene function will be verified using gene knock-out and recovery techniques, followed by functional verification on the biosynthesis of toxins or pigments.

植物病原真菌的聚酮体合成酶（PKS）主要参与毒素、色素及6-甲基水杨酸的生物合成，而目前芦笋茎枯病菌PKS基因pks-pa的功能尚不明确。在前期研究中，本课题组首先用PCR方法对芦笋茎枯病菌PKS的类型进行了鉴定，确定为WA型；用特异性及简并引物进行PCR扩增，获取了部分片段。本项目拟在前期研究的基础上，首先通过5'/3'-RACE及Tail-PCR技术获取cDNA及gDNA全长序列，并通过公共基因数据库和相关软件对该基因进行生物信息学分析。然后，构建芦笋茎枯病菌pks-pa基因在大肠杆菌中的表达载体，并通过工程菌筛选与验证获得诱导表达的阳性菌株；通过重组蛋白的可溶性、Western blotting等分析，明确芦笋茎枯病菌PKS的蛋白属性。最后，构建该病菌pks-pa基因敲除表达载体，用基因敲除与回复技术鉴定该基因的功能，通过对突变体的表型测定，明确该基因究竟参与毒素还是色素的生物合成。

通过PCR检测，明确了芦笋茎枯病菌聚酮体合成酶属于WA型。通过RT-PCR获取了芦笋茎枯病菌pks-pa基因的cDNA片段。分别设计了3'/5’-RACE的相关引物，获取3'端和5'端的cDNA序列，获取了该病菌pks-pa基因cDNA全长序列。用PCR方法扩增pks-pa基因的gDNA部分片段，通过TAIL-PCR扩增了两端的侧翼序列并进行拼接，获得了gDNA全长序列。用PCR扩增pks-pa基因的DNA序列，将PCR产物切胶回收后，克隆到pMD20-T载体，将双酶切后的pks-pa基因片段和表达载体pET-30a(+)大片段，T4-DNA 连接酶16℃连接过夜，获得表达载体pET-30a(+)-pks-pa（连接产物）。连接产物通过CaCl2法转化E. coli DH5α感受态细胞，经卡那霉素(Kan) 100μg/mL、蓝白斑初步筛选，然后通过菌落PCR和EcoR I/Xho I双酶切筛选和鉴定阳性重组质粒。随机挑取了3个含重组表达质粒的大肠杆菌DH5α单菌落，通过10% SDS-PAGE、考马斯亮蓝染色确定其表达情况。以芦笋茎枯病菌野生型XT2菌株基因组DNA 为模板，用PKSF1/R1引物对扩增pks-pa 基因上游的1093 bp的 5'同源臂片段进行扩增。用PKSF2/R2引物对扩增1018bp左右的 pks-pa基因下游3'同源臂片段进行扩增。将上游5'和下游3'同源臂片段之相连转化最终得到正确的pks-pa基因敲除质粒载体mypb21。以芦笋茎枯病菌XT2菌株基因组DNA为模板用引物pksF/R扩增含pks-pa完整基因及其部分旁侧序列的片段，将PCR产物纯化后直接TA克隆到pMD18-T载体中，最终得到回复载体pMD18-pks-pa。通过原生质体转化，使G418抗性基因取代pks-pa基因，获得敲除菌株XT2-knock。回复突变筛选标记用带有潮霉素B抗性基因的pUCATPH 共转化载体。分别用EcoR I 酶切线性化回复载体pMD18-βNAC 和筛选标记载体pUCATPH，将其共同转化到突变株XT2-knock原生质体中，获得回复菌株XT2-pks-pa。通过对野生型菌株XT2、敲除菌株XT2-knock和回复菌株XT2-pks-pa生物学及致病力比较，明确了pks-pa与致病力无相关性，而与色素产生具有正相关性。