目录
摘 要 II
关键词 II
Abstract III
引言
引言 1
1 材料与方法 2
1．1 实验材料 2
1．1．1 供试样品 2
1．1．2 实验仪器设备 2
1．1．3 培养基和试剂 2
1．2 实验方法 2
1．2．1 筛选并分离纯化硝磺草酮抗性菌株 2
1．2．2 抗性菌株16S rRNA基因序列测定及系统发育学分析 2
1．2．3 供试菌株硝磺草酮抗性检测 3
1．2．4 硝磺草酮抗性基因的克隆 3
1．2．5 HPPD基因的表达与蛋白纯化 4
1．2．6 HPPD酶活检测 5
1．2．7 菌种保藏 6
2 结果与分析 6
2．1 硝磺草酮抗性菌的筛选和分离纯化 6
2．2 16S rRNA基因序列扩增和同源发育分析 7
2．3 供试菌株硝磺草酮抗性检测 10
2．4 HPPD基因克隆和同源性分析 12
2．5 HPPD外源表达 13
2．6 XaHPPD酶活检测 14
3 讨论 14
致谢 15
参考文献 16
硝磺草酮抗性菌株的筛选和抗性基因的克隆表达
摘 要
硝磺草酮是一种对羟苯基丙酮酸双加氧酶(HPPD)抑制型除草剂，筛选硝磺草酮抗性菌株并克隆表达抗性基因，可以为研发硝磺草酮抗性作物提供基因资源。以硝磺草酮为选择压力筛选抗性菌株，革兰氏染色，PCR扩增16S rRNA基因序列，系统发育分析初步鉴定目的菌株，检验抗性。克隆高抗性菌株HPPD基因，构建表达载体，转化至大肠杆菌中表达，Co2+亲和层析后透析纯化蛋白产物，采用酶偶联法和高效液相色谱法分别定性和定量分析产物酶活性。本实验在2000 mg/L硝磺草酮TMSM选择培养基中分离出具有硝磺草酮抗性的革兰氏阴性菌共6株，4株为假单胞菌，1株为硫氧化杆菌，1株为黄单胞菌，其中Xanthomonas sp.  *51今日免费论文网|www.51jrft.com +Q: ^351916072# 
C8对硝磺草酮的抗性最强，最高耐受4000 mg/L硝磺草酮。克隆C8的HPPD基因并命名为Xahppd，大小为996 bp，GC含量63.45%，编码332个氨基酸，BLAST分析发现XaHPPD与Cupriavidus taiwanensis中蛋白质CBM2588_B90063氨基酸序列相似性最高，达到49.07 %。Xahppd在大肠杆菌中表达，蛋白产物分子量大小约为40 kDa，纯化后产物具有HPPD活性，比酶活为9.7 U/mg。基因Xahppd具有硝磺草酮抗性，且外源表达产物有HPPD活性，在HPPD抑制剂抗性转基因作物的培育中具备一定的应用价值。
Isolation of mesotrioneresistant strain and cloning and expression of hppd gene
Abstract
Mesotrione is an HPPDinhibiting herbicide with good effect and high security. Screening mesotrioneresistant strain, cloning and expressing hppd gene can provide genetic resources for the construction of mesotrioneresistant crops. Selecting resistant strains by mesotrione, the target strains are identified from resistant strains by phylogenetic analysis after gram staining and PCR amplification of 16S rRNA genetic sequence. The gene hppd was cloned from the resistant strain by PCR, and the amplified products was expressed in Escherichia coli BL21(DE3). The protein product was purified by Co2+ affinity chromatography and dialysis in phosphate buffered saline. The enzyme activity was qualitatively and quantitatively analyzed using the enzymecoupled assay and HPLC. Six strains of gram negative bacteria with mesotrione resistance were isolated, four strains are Pseudomonas sp., one strain is Ciceribacter sp., and one strain is Xanthomonas sp.. Xanthomonas sp. C8 has the best resistance to mesotrione, with the tolerance of 4000 mg/L mesotrione. The gene Xahppd was cloned from Xanthomonas sp. C8, with the length of 996 bp and the GC content of 63.45%, which encods 332 amino acids, showed that XaHPPD has the highest identity (49.07%) with the protein CBM2588_B90063 of Cupriavidus taiwanensis. Xahppd was expressed in E. coli and the molecular mass of the product was about 40 kDa. After purification and activity analysis, the product XaHPPD activity reached 9.7 U/mg. In this study, a high mesotrioneresistant bacteria Xanthomonas sp. C8 was isolated and an HPPD gene Xahppd was cloned. XaHPPD shows phydroxyphenylpyruvate dioxygenase activity, which indicates that this gene is useful in mesotrione resistant transgenic engineering.

原文链接：http://www.jxszl.com/swgc/smkx/606544.html