稻曲病菌T-DNA插入突变体B2510的插入位点分析

doi:10.16819/j.1001-7216.2016.5198

摘要/Abstract

摘要：

以稻曲病菌T-DNA插入突变体库中致病力减弱突变菌株B2510为材料,通过分析T-DNA插入位点的侧翼序列和突变基因,分离出在稻曲病菌致病过程中起作用的基因。通过测定突变菌株B2510的生长速率、产孢能力及致病力发现,与野生型菌株P1相比,B2510田间接种表现为致病性减弱;在MM培养基上生长速率下降,而在PSA和TB₃培养基中生长速率与野生型没有显著差异,但丧失产孢能力。Southern杂交显示T-DNA在突变菌株B2510中以双拷贝形式插入,利用TAIL-PCR技术扩增紧邻T-DNA两侧的侧翼序列,经过比对分析发现,T-DNA分别插在基因UV8b_1412的启动子区域和UV8b_1386的下游3'端,且稻曲菌基因组序列均未丢失,T-DNA上只有几个碱基发生变化。半定量RT-PCR分析基因的表达情况,显示两个基因在突变体B2510的表达量较P1均显著下降,推测T-DNA插入位点处的基因与稻曲病菌致病性相关,可能在某一阶段参与调控稻曲病菌在水稻上的致病过程。

关键词: 稻曲病菌, T-DNA插入突变, ATMT转化, 致病力, 侧翼序列

Abstract:

To isolate the gene concerned with molecular pathogenic process of Ustilaginoidea virens (U. virens) , T-DNA integration flanking sequence and the mutant genes of a mutant strain B2510 were analyzed. Compared with the wild-type U. virens strain P1, the pathogenicity of the mutant strain B2510 in the field was significantly decreased. The growth rate of B2510 on MM medium was slower than that of P1, but was not significantly different on PSA and TB₃ medium which were nutritionally endowed. The mutant strain B2510 did not produce conidiophores in PS broth medium. Genomic Southern bolt analysis confirmed that there were double T-DNA events inserted in genome of mutant strain B2510. The flanking U. virens sequences of T-DNA obtained by TAIL-PCR were adjacent in the wild type and with no sequences lost, and only a few bases of T-DNA were changed. The T-DNA insertion site was in the promoter region of UV8b_1412 and downstream 3'region of UV8b_1386, respectively. RT-PCR analysis confirmed that the expression of both of two genes in B2510 were significantly decreased. The genes which were affected by T-DNA insertion may be associated with pathogenicity and participate in the regulation of pathogenic process of U. virens in rice.

Key words: Ustilaginoidea virens, T-DNA insertion mutant, ATMT transformation, pathogenicity, flanking sequence

中图分类号:

丁慧, 俞咪娜, 王亚会, 于俊杰, 尹小乐, 薄惠文, 黄星, 刘永锋. 稻曲病菌T-DNA插入突变体B2510的插入位点分析[J]. 中国水稻科学, 2016, 30(5): 541-551.

Hui DING, Mi-na YU, Ya-hui WANG, Jun-jie YU, Xiao-le YIN, Hui-wen BO, Xing HUANG, Yong-feng LIU. Molecular Characterization of Ustilaginoidea virens T-DNA Insertion Mutant B2510[J]. Chinese Journal OF Rice Science, 2016, 30(5): 541-551.

图/表 7

表1 引物列表

Table 1 Primers applied in the research.

引物名称 Name	引物序列 Sequence(5’ → 3’)
hyg1.4F^[21]	ACAGAAGATGATATTGAAGGAGC
hyg1.4R^[21]	TACTCTATTCCTTTGCCCTCG
LAD8^[21]	ACGATGGACTCCAGAGCGGCCGCVVNVNNNACGTG^*
LAD10^[21]	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT^*
GFP-1^[21]	CCATCCTGGTCGAGCTGGA
GFP-2^[21]	CGAACTCCAGCAGGACCATG
GRB3^[21]	TGGGCACCACCCCGGTGAACAGCTC
HLB1^[21]	GATTCCCAATACGAGGTCGCCAA
HLB2^[21]	TGGAGGCCGTGGTTGGCTTGTATGGAG
HLB3^[21]	AGGGTCGATGCGACGCAATCGTCCGA
AC-S2^[21]	GAGTTTAGGTCCAGCGTCCGTCGACGATGGACTCCAGAG
AC-S3^[21]	GAGTTTAGGTCCAGCGTCCGT
8H1	CGAGTTCCAACAGACCCCTA
8R1	ATGGCCAAACTTCAACCCTT
10H1	ATGGACCAGCCACAACTAAA
10R1	TGTCAAAGATGCAAGGTCA
RT-1F	GACCTTTTGTTCCGAGCCAT
RT-1R	GTCTTTGGCGGGTTTATCAG
RT-U-1	ACCGCTTCAGCAGATGGG
RT-U-2	GGCGTCTGCTACTCGCTCTA
RT-D-1	GTCCCCTCAGGTCGCAATAG
RT-D-2	TAAGTTCCCTCCCACCAGCC
α-tubulin2F^[24]	GGCGTTTACAATGGCACTTC
α-tubulin2R	CGGAACAGTTGACCAAAAGG

图1 稻曲病菌突变菌株B2510的分子检测 A-PCR检测,其中泳道1、3分别为以P1为模板检测GFP基因和HPH基因; 泳道2、4分别为以B2510为模板检测GFP基因和HPH基因。B-Southern杂交检测,其中泳道1为P1基因组Sal Ⅰ、Hind Ⅲ双酶切后HPH基因Southern杂交;泳道2、3分别为B2510基因组Sal Ⅰ、HindⅢ酶切后HPH基因Southern杂交。

Fig. 1. Molecular detection of mutant strain B2510. A, PCR analysis of the GFP gene and HPH gene. Lanes 1 and 2, PCR detection of the GFP genes of P1 and B2510, respectively. Lanes 3 and 4, PCR detection of the HPH of P1 and B2510. Lane M, DNA marker; B, Southern blot detection of B2510, showing that a double T-DNA insertion event occurred in the B2510 genome. Lane 1, Genomic DNA of P1 was digested with Sal Ⅰ and Hind Ⅲ. Lanes 2 and 3, Genomic DNA of B2510 was digested with Sal Ⅰ, Hin dⅢ, respectively.

图2 突变菌株B2510的致病性测定 A-突变菌株B2510接菌稻穗上的稻曲球数显著少于野生菌株P1。粗箭头所指为稻曲球; B-突变菌株B2510和野生菌株P1分别接种两优培九后病粒数统计。**表示极显著性差异,P < 0.05。

Fig. 2. Pathogenicity test of the mutant strain B2510. A,Number of diseased grains caused by the mutant strain B2510 was significantly decreased than that of wild strain P1. The coarse black arrows indicate false smut balls; B, Statistical analysis of the number of diseased grains per panicle on Liangyoupeijiu. ** shows significant difference at 0.05 level.

图3 突变菌株B2510和野生菌株P1的菌丝生长及产孢特性分析 A-突变菌株B2510与野生菌株P1在MM、PSA 和TB3这三种不同培养基上菌落直径的比较。在MM培养基中突变菌株B2510的生长速率明显慢于P1,且差异极显著;B-突变菌株B2510与P1分别在MM、PSA 和TB3不同培养基上的菌落形态, 图中标尺=1 cm; C-在显微镜(10× 40)下观察到的突变菌株B2510与P1孢子和菌丝,标尺= 20 μm;D-突变菌株B2510与P1菌丝直径的比较。

Fig. 3. Analysis of colonies and sporulation of mutant strain B2510 and wild-type strain P1. A, Difference of colony diameters between mutant strain B2510 and wild-type strain P1 on MM, PSA and TB3 medium. The growth rate of B2510 was significantly slower than that of P1; B, Colonies of mutant strain B2510 and P1 on different media; bar=1 cm; C, Analysis and comparison of conidium and hypha between mutant strain B2510 and P1 under microscope (10× 40); bar= 20 μm; D, Comparison of the sizes of hypha between mutant strain B2510 and P1.

图4 突变菌株B2510 T-DNA 插入位点侧翼序列的扩增与分析 A-突变体B2510中T-DNA插入的结构以及各引物在基因组上的位置; B,C-PCR验证T-DNA插入位点处右端的侧翼序列,并分析T-DNA插入后的基因组序列变化。

Fig. 4. Cloning and analysis of T-DNA flanking region in mutant strain B2510. A, Schematic diagram of T-DNA insertion event in mutant B2510 and the positions of primers on genome; B and C,Analysis of T-DNA right border flanking region and genome sequence after T-DNA insertion using PCR.

图5 稻曲病菌突变菌株B2510中T-DNA 插入位点侧翼基因分析 A,B-两个基因中 T-DNA插入位置; C,D-RT-PCR检测突变菌株B2510中两个基因的表达变化,α-tubulin为内参基因。D中Uv8b_1386-Up指的是该基因5'端的表达量,Uv8b_1386-Down则是基因3'端的表达量。

Fig. 5. Analysis of T-DNA flanking gene in U.virens mutant strain B2510. A and B, Schematic diagram of T-DNA insertion event in the two genes; C and D, RT-PCR analysis of the expression level of the two genes in mutant strain B2510, α-tubulin was used as the reference gene. In Fig.5-D, Uv8b_1386-Up indicates the expression of 5' flanking region of Uv8b_1386, and Uv8b_1386-Down, the expression of the gene 3' flanking region.

图6 基于蛋白氨基酸序列的稻曲病菌及其他多种真菌的系统发育分析 A-通过MEGA6程序计算其分散距离,制作稻曲病菌的C6 转录因子与其他16种不同真菌同源蛋白的系统发育树;每个菌株后的号码与GenBank中登记的一致。B-通过MEGA6程序计算其分散距离,制作稻曲病菌的类RasGTP酶激活蛋白与其他10种不同真菌同源蛋白的系统发育树;每个菌株后的号码与GenBank中登记的一致。

Fig. 6. Phylogenetic tree of U.virens and several fungi based on amino acid sequence of proteins. A, Phylogenetic tree of C6 transcription factor of U.virens and 16 other species was constructed by observed divergency distance method in the program MEGA6. Numbers correspond to GenBank accession numbers. B, Phylogenetic tree of GTPase-activator protein for Ras-like GTPase containing protein of U.virens and 10 other species was constructed by observed divergency distance method in the program MEGA6. Numbers correspond to GenBank accession numbers.

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