稻曲病菌T-DNA插入突变体B1464插入位点分析

doi:10．3969/j．issn．1001G7216．2015．03．01

摘要/Abstract

摘要：

以稻曲病菌致病力丧失的突变菌株B1464为材料,对其生物学形态和分子遗传变异进行分析。B1464田间接种表现为不致病,与野生型菌株相比, 在营养贫瘠的MM培养基上生长速率没有显著差异,而在PSA和TB3培养基中生长速率较慢,并且在PS液体培养基中产孢能力下降,同时突变菌株的分生孢子与野生型相比形态和大小均发生变化。Southern杂交结果显示T-DNA在突变菌株B1464中以单拷贝形式插入,利用TAIL-PCR技术扩增得到的紧邻T-DNA两侧的侧翼序列在野生型中不相邻,与野生型菌株基因组比对发现突变菌株插入位点处丢失约20 kb的DNA片段。RT-PCR结果表明突变菌株中T-DNA插入位点两侧基因表达量均下调。推断突变菌株B1464的致病能力丧失可能是由于T-DNA的插入导致了染色体重排及破坏了某些基因的正常表达。

关键词: 稻曲病菌, T-DNA插入突变, 致病力, 苹果酸合成酶, 铜转运蛋白

Abstract:

With an avirulent Ustilaginoidea virens strain B1464 as material,we analyzed the changes in biological morphology and pathogenic mechanism. Compared to the wild-type U. virens strain P1, the mutant strain B1464 showed no pathogenicity in the field. The rate of growth on MM medium was not different to P1 and B1464. On PSA and TB3 medium the rates of growth were nutritionally endowed. B1464 grew slower and produced few conidiophores in PS medium, and its conidiophores morphology and spore size became smaller. Genomic Southern bolt analysis confirmed that B1464 was single T-DNA insertional events. The flanking sequences of T-DNA obtained by TAIL-PCR were non-adjacent in the wild type. It was founds that the mutant strain lost about 20 kb sequences in the insertion site comparing with the wild type strain. The expression of the flanking genes of the T-DNA was down-regulated, detecting by semi-quantitative RT-PCR. The reason of the chromosome rearrangement and abnormal expression of some genes is the insertion of T-DNA, probably, leading to the mutant strain B1464 lost its pathogenicity.

Key words: Ustilaginoidea virens, T-DNA insertion mutant, pathogenicity, malate synthase, copper transporter

中图分类号:

王亚会, 刘永锋, 陆凡, 俞咪娜, 黄磊, 郑梦婷, 于俊杰, 尹小乐. 稻曲病菌T-DNA插入突变体B1464插入位点分析[J]. 中国水稻科学, 2015, 29(3): 311-318.

Ya-hui WANG, Yong-feng LIU, Fan LU, Mi-na YU, Lei HUANG, Meng-ting ZHENG, Jun-jie YU, Xiao-le YIN. Molecular Characterization of T-DNA Integration into Ustilaginoidea virens Mutant B1464[J]. Chinese Journal OF Rice Science, 2015, 29(3): 311-318.

图/表 6

表1 本研究所用的引物信息

Table 1 Primers applied in the research.

引物名称 Name	引物序列 Sequence(5' → 3')
hyg1.4F	ACAGAAGATGATATTGAAGGAGC
hyg1.4R	TACTCTATTCCTTTGCCCTCG
LAD10	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT^*
GFP-1	CCATCCTGGTCGAGCTGGA
GFP-2	CGAACTCCAGCAGGACCATG
GLB1	AAGATGGTGCGCTCCTGGACGTAG
GLB2	ACGATGGACTCCAGAGGCACTGCACGCCGTAGGTCAGGGTGG
GLB3	TGGGCACCACCCCGGTGAACAGCTC
HLB1	GATTCCCAATACGAGGTCGCCAA
HLB2	TGGAGGCCGTGGTTGGCTTGTATGGAG
HLB3	AGGGTCGATGCGACGCAATCGTCCGA
AC-S2	GAGTTTAGGTCCAGCGTCCGTCGACGATGGACTCCAGAG
AC-S3	GAGTTTAGGTCCAGCGTCCGT
G-1	TGGGATTGAGCGGGAGCT
H-1	GGCTGCTGTACGACGAGATC
U-1	CCAGTAGACGCAAGCACAGG
G-2	CACCACGACCACCGTCTCCT
G-3	CTGTACGCAAGCTCGTGGAC
H-2	CGAGGCGGAACTTGTTGTGC
H-3	GCTGCGTGCCCATCAACTAC
β-actin F^[22]	CCGTGAGAAGATGACCCAGA
β-actin R	GGCGAAACCCTCGTAGATGG

图1 稻曲病菌TDNA插入突变体B1464的分子检测(A-PCR检测; 1和3分别指以P1为模板检测gfp基因和HPH基因; 2和4分别指以B1464为模板检测gfp基因和HPH基因;M-DNA标记。B-Southern杂交检测; 1-P1基因组EcoRⅠ、Hind Ⅲ酶切后HPH基因Southern杂交; 2、3分别为B-1464基因组EcoR Ⅰ、HindⅢ酶切后HPH基因Southern杂交。)

Fig.1. Molecular detection of Ustilaginoidea virens mutant B1464 strain. (A, PCR analysis of the gfp gene and HPH gene. Lanes 1 and 2, PCR detection of the gfp genes of P1 and B1464, respectively. Lanes 3 and 4, PCR detection of the HPH of P1 and B1464. Lane M, DNA marker. B, Southern blot detection of B1464, showing that a single T-DNA insertion event occurred in the B1464 genome. Lane 1, Genomic DNA of P1 was digested with EcoR Ⅰ, Hind Ⅲ. Lanes 2 and 3, Genomic DNA of B1464 was digested with EcoRⅠ,Hind Ⅲ, respectively.)

图2 稻曲病菌T-DNA插入突变体B1464表型分析(A-菌株在TB3、PSA和MM培养基上的菌落直径; **代表B1464与P1间差异达0.01显著水平; B-不同培养基上B1464与P1的菌落形态; C-B1464和P1的孢子形态; D-菌株孢子大小比较。)

Fig. 2. Analysis of the phenotypie of Ustilaginoidea virens mutant B1464. (A, Colony diameters on TB3, PSA and MM medium; **indicate significant difference between B1464 and P1 at 0.01 level; B, Colonies of B1464 and P1 on different media; C, Conidium morphology of B1464 and P1; D, Sizes of conidium.)

图3 T-DNA结构及插入位点侧翼序列(A-T-DNA结构;B-T-DNA的RB结构和侧翼序列,加下划线部分片段在T-DNA和稻曲基因组中均未检测到; C-T-DNA的LB结构和侧翼序列。)

Fig. 3. Structure of T-DNA and flanking sequence. (A, The structure of T-DNA; B, Structure of T-DNA RB and flanking sequence, the underlined fragments were not found in the genome of U.virens and T-DNA; C, Structure of T-DNA LB and flanking sequence.)

图4 对B1464 T-DNA插入位点存在染色体重排现象的验证(A, Insertion site to B1464 and primer design;B, PCR detection. )

Fig. 4. Verification of the molecular characterization of the chromosomal rearrangement in B1464. (A, Insertion site to B1464 and primer design;B, PCR detection. )

图5 B1464中两端基因的表达变化的RT-PCR检测(A-B1464中存在序列缺失及插入位点两侧基因;B-RT-PCR检测B1464中两基因表达变化。)

Fig.5. RT-PCR analysis of the expression level of the two genes in B1464. (A, Loss of sequence and the two flanking genes in B1464; B, RT-PCR analysis of the expression level of the two genes in B1464. )

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