<i>Folpcs1</i>基因对尖孢镰刀菌亚麻专化型的无性繁殖和营养生长的调控 <sup>*</sup>

doi:10.13523/j.cb.1908059

中国生物工程杂志

2020, Vol. 40

Issue (3): 48-64 DOI: 10.13523/j.cb.1908059

研究报告

Folpcs1基因对尖孢镰刀菌亚麻专化型的无性繁殖和营养生长的调控 ^*

郭晶,侯占铭(

)

内蒙古师范大学生物科学与技术学院呼和浩特 010022

Folpcs1 Is Responsible for Asexual Reproduction and Vegetative Growth in Fusarium oxysporum f. sp. Lini.

GUO Jing,HOU Zhan-ming(

)

College of Life Sciences and Technology, Inner Mongolia Normal University, Hohhot 010022, China

全文: PDF(4334 KB) HTML

摘要：

背景尖孢镰刀菌亚麻专化型[Fusarium oxysporum Schl.f.sp.Lini(Bolley) Snyder & Hansen]是一种引起亚麻枯萎病的土传真菌,对亚麻的产量及质量有严重的危害.研究表明,在小麦赤霉菌中C2H2型锌指转录因子Pcs1调控中间瓶梗(intercalary phialides)产生分生孢子.目的:鉴定pcs1同源基因Folpcs1在镰刀菌中的作用.方法: 对Folpcs1的基因组DNA及cDNA测序后,利用Split-Marker 基因敲除技术破坏该基因.构建含有潮霉素抗性基因(hph)的基因缺失盒.通过聚乙二醇(PEG)转化野生型原生质体法,获得了Folpcs1基因缺失突变株(ΔFolpcs1),并利用PCR法进行正负筛选.为了对缺失突变体互补,将pcs1及其上下游侧翼序列构建到pZWH1中后,回复到ΔFolpcs1中.结果: 测序结果表明,有一个654bp的内含子,cDNA序列长度为2 846bp.通过观察发现,敲除突变体的生长速率明显降低,培养基中几乎观察不到分生孢子.回复突变体回复了野生型菌株的生长速率及分生孢子产生过程.结论: 说明Folpcs1负责菌丝的营养生长和分生孢子的产生.

关键词： 尖孢镰刀菌亚麻专化型; Folpcs1; Split-Marker; 基因敲除

Abstract:

Fusarium oxysporum f.sp.Lini, a forma specialis of Fusarium oxysporum on flax, is soil-borne fungus which causes Fusarium wilt of flax, a serious disease of flax resulting in great loss of yield and quality of the crop. It has been identified that C2H2 zinc finger transcription factor Pcs1 in Fusarium graminearum is responsible for the generation of conidia produced from intercalary phialides on hyphae. Objective:The gene disruption of the Folpcs1 is performed according the principle of homologous recombination for identifying the function of the Folpcs1, the homologous gene of the pcs1, in Fusarium oxysporum f. sp. Lini. Methods:The disruption of the gene was made by the Split-Marker strategy after the genomic and complementary DNA was sequenced. The deletion cassettes containing a hygromycin resistance gene (hph) was constructed and transformed into protoplast of the wild type of the fungus mediated by polyethylene glycol. The deletion mutants, ΔFolpcs1s, were obtained and confirmed by PCR using positive and negative primer. To make complementation for the deletion mutuant, the coding frame sequence of the Folpcs1 gene, together with its upstream and downstream flanking sequence, was cloned into the pZWH1 containing neomycin resistant gene and transformed into the deletion mutant. Results:The sequencing revealed that the gene contained one intron of 654bp and the whole cDNA sequence consisted of 2 846bp. The morphological and microscopic observation found that growth rate of deletion mutant significantly reduced and the conidium was hardly observed in culture mixture of the mutant. The revertant recovered in normal growth and conidiogenesis of the wild type. Conclusion:The results suggest that Folpcs1 is responsible for asexual reproduction and vegetative growth in Fusarium oxysporum f. sp. Lini.

Key words: Fusarium oxysporum Schl.f.sp.Lini Folpcs1 Split-marker Gene disruption

收稿日期: 2019-08-29 出版日期: 2020-04-18

ZTFLH:

Q78

基金资助: * 国家自然科学基金(31160280);内蒙古自治区自然科学基金(2015MS0311)资助项目

通讯作者: 侯占铭 E-mail: houzhm@imnu.edu.cn

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引用本文:

郭晶,侯占铭. Folpcs1基因对尖孢镰刀菌亚麻专化型的无性繁殖和营养生长的调控 ^*[J]. 中国生物工程杂志, 2020, 40(3): 48-64.

GUO Jing,HOU Zhan-ming. Folpcs1 Is Responsible for Asexual Reproduction and Vegetative Growth in Fusarium oxysporum f. sp. Lini.. China Biotechnology, 2020, 40(3): 48-64.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1908059 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I3/48

表1 pMD19-T载体克隆体系

表2 引物序列

表3 一步法无缝定向克隆体系

图1 尖孢镰刀菌亚麻专化型基因组DNA及RNA

图2 Folpcs1基因cDNA末端克隆

图3 Folpcs1 DNA及cDNA的获得

图4 四种真菌氨基酸序列比对

图5 根据10种真菌氨基酸序列构建的系统进化树

图6 Folpcs1 基因缺失盒的构建

图7 Folpcs1 转化子的筛选与鉴定

图8 不同菌株表型分析

表4 不同菌株中分生孢子的产量

图10 Folpcs1基因及其上下游侧翼序列片段PCR产物

图11 重组质粒的pZGH1的筛选

图12 回复突变体Folpcs1-C的PCR筛选

图13 不同菌株的表型分析

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