球孢白僵菌四个Septin基因的功能解析与无外源抗性标记表达杀虫蛋白Vip3Aal的工程菌构建

发布时间：2018-01-21 09:48

本文关键词： 球孢白僵菌 septin基因家族基因敲除萌发产孢多胁迫应答毒力转基因技术乳清苷5-磷酸脱羧酶基因ura3 营养期杀虫蛋白Vip3A ELISA 生防潜能　出处：《浙江大学》2016年硕士论文　论文类型：学位论文

【摘要】：球孢白僵菌(Beauveria bassiana)是重要的昆虫病原真菌,目前广泛应用于农林害虫的生物防治。其制剂的有效成份为活体分生孢子,田间杀虫效果的稳定性及持效性受作物环境或季节性因素的制约,如环境温度、紫外线辐射、化学农药等。因此,研究发掘球孢白僵菌生长发育及抗逆性状的调控基因服务于菌株的遗传改良,具有重要的现实意义。同时,通过基因工程技术在提高生防真菌生防潜能的同时尽可能降低环境安全性风险,是需要探索的重要技术难题。因此,本论文开展了两方面的研究工作,一是研究解析了球孢白僵菌Septin基因家族的基本生物学功能,二是探索了利用内源乳清酸核苷5-磷酸脱羧酶基因ura3作为营养缺陷型安全性标记,在球孢白僵菌中表达昆虫中肠特异性胃毒蛋白Vip3Aa1而构建低风险工程菌株的可能性。球孢白僵菌Septin基因家族的功能解析Septin是在进化上高度保守的一类GTP结合蛋白,在结构上与Ras GTP激酶家族的成员相似。在球孢白僵菌中存在4个Septin蛋白(BspA/B/C/D),与12种代表性丝状真菌中对应蛋白的同源性达到79%以上。Septin基因的缺失在不同程度上影响球孢白僵菌的萌发、生长及分生孢子形成。敲除株的分生孢子萌发提速,但菌丝细胞隔膜形成滞后且数量减少,以致生长受到抑制,尤在果糖作为唯一碳源的基础培养基上表现较严重的生长缺陷。在标准培养基上正常培养期间,分生孢子产量在敲除株ΔbspA中下降80～89%,在ΔbspB中下降49～67%,在ΔbspC中下降60～76%,在ΔbspD中下降10～39%。在胁迫应答测定中,敲除株在生长期间对胞壁干扰剂刚果红的敏感性升高,而对强氧化剂及高渗剂的敏感性则有所降低。其分生孢子耐高温和紫外辐射能力也有所变化。其中,ΔbspA耐高温、耐紫外辐射能力均有所下降,ΔbspD仅表现耐紫外辐射的能力下降,而ΔbspB耐高温能力增强,ΔbspC耐紫外辐射能力提高。但是,敲除株对大蜡螟幼虫的毒力与野生株和回补株相比未见有实质性的变化。结果表明,4个septin蛋白不仅是球孢白僵菌萌发生长、细胞分隔、产孢的重要调控因子,还参与多胁迫应答反应的调控,但与毒力无显著关联。利用内源安全标记基因构建无外源抗性标记的球孢白僵菌工程菌株乳清酸核苷5-磷酸脱羧酶在细胞内可催化尿苷(尿嘧啶)的合成,参与基因的翻译而影响细胞生长。其基因ura3缺失致使细胞自身不能有效合成尿苷或尿嘧啶,因而无法正常生长,但加入外源尿苷即可恢复生长。利用这一特性,以球孢白僵菌ura3作为营养缺陷型标记取代原有的抗除草剂基因草丁膦抗性基因bar,进而构建出表达昆虫中肠特异性胃毒蛋白Vip3Aa1的一系列工程株BbHUV,经PCR、qRT-PCR及ELISA检测,证明多数转化子的菌丝和分生孢子中都有目标杀虫基因或蛋白的表达,由此建立起无外源风险抗性标记的工程菌构建平台。
[Abstract]:Beauveria bassiana (Beauveria bassiana) is an important entomopathogenic fungus, which is widely used in the biological control of agricultural and forestry pests. The stability and efficacy of field insecticidal effects are restricted by crop environment or seasonal factors, such as ambient temperature, ultraviolet radiation, chemical pesticides and so on. It is of great practical significance to explore the regulatory genes of Beauveria bassiana growth and resistance to stress for the genetic improvement of the strain. It is an important technical problem to improve the biocontrol potential of fungi and reduce the risk of environmental safety by genetic engineering. Therefore, two aspects of research work have been carried out in this paper. First, the basic biological functions of Septin gene family of Beauveria bassiana were analyzed. The second is to explore the use of endogenous nucleoside 5-phosphate decarboxylase gene ura3 as a nutritional deficiency safety marker. It is possible to construct a low-risk engineering strain by expressing insect midgut specific gastrotoxic protein Vip3Aa1 in Beauveria bassiana. The functional analysis of the Septin gene family of Beauveria bassiana is expressed in the gene family of Beauveria bassiana. A highly conserved class of GTP binding proteins. The structure is similar to that of Ras GTP kinase family. There are four Septin proteins BspA / B / C / D in Beauveria bassiana. The homology of the corresponding protein with 12 representative filamentous fungi was above 79%. The deletion of Septin gene affected the germination of Beauveria bassiana to varying degrees. Growth and conidial formation. The conidial germination of knockout strain increased, but the formation of hyphal cell membrane was delayed and the number decreased, resulting in the inhibition of growth. Especially in the basal medium with fructose as the sole carbon source, the growth defects were more serious. During the normal culture on the standard medium, the conidial yield decreased by 80% to 89% in the knockout plant 螖 bspA. The contents of 螖 bspB, 螖 bspC, 螖 bspD and 螖 bspD were decreased by 49%, 60% and 1039%, respectively. The sensitivity of the knockout strain to the cell wall interferential agent Congo red increased, but to the strong oxidant and hyperosmotic agent decreased, and its conidial resistance to high temperature and ultraviolet radiation also changed. The ability of 螖 bspA to resist high temperature and ultraviolet radiation were all decreased. 螖 bspD only showed the ability of ultraviolet radiation resistance decreased, while 螖 bspB's ability to resist high temperature increased. The ability of 螖 bspC to resist ultraviolet radiation was improved. However, the virulence of knockout plant to larva was not significantly different from that of wild plant and compensatory plant. The results showed that there was no significant change in the virulence of the knockout plant. The four septin proteins were not only important regulatory factors for germination and growth of Beauveria bassiana, but also involved in the regulation of response to multiple stress. However, there was no significant correlation with virulence. The nucleoside 5-phosphate decarboxylase of whey nucleoside 5-phosphate could catalyze the synthesis of uridine (uracil) in cells. Participate in the translation of genes and affect cell growth. The deletion of the gene ura3 makes the cells can not effectively synthesize uridine or uracil, so they can not grow normally. However, exogenous uridine could restore growth. By using this characteristic, the herbicide resistant gene bar was replaced by ura3 as a nutritional deficiency marker. Then a series of engineering strain BbHUVexpressing insect midgut specific gastrotoxic protein Vip3Aa1 were constructed and detected by PCR qRT-PCR and ELISA. It was proved that the target insecticidal gene or protein was expressed in the hyphae and conidium of most transformants, and the construction platform of engineering bacteria without exogenous risk resistance markers was established.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476

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