木薯UDP依赖型糖基转移酶基因<i>MeUGT25</i>在抗细菌性枯萎病中的功能研究

曾坚; 陈颍桐; 蔡美琪; 李丽珍; 林墁; 刘思雯; 刘博婷; 胡伟; 曾力旺

doi:10.19303/j.issn.1008-0384.2023.12.009

木薯UDP依赖型糖基转移酶基因MeUGT25在抗细菌性枯萎病中的功能研究

doi: 10.19303/j.issn.1008-0384.2023.12.009

曾坚^1,,
陈颍桐¹,
蔡美琪¹,
李丽珍¹,
林墁¹,
刘思雯^{1, 3},
刘博婷¹,
胡伟²,
曾力旺^2, ,

1.
韶关学院广东省粤北食药资源利用与保护重点实验室/韶关学院生物与农业学院，广东　韶关　512005
2.
中国热带农业科学院热带生物技术研究所，海南　海口　571101
3.
华南农业大学亚热带农业生物资源保护与利用国家重点实验室，广东　广州　510642

基金项目: 广东省基础与应用基础研究基金（2023A1515010336、2021A1515011236）；广东省普通高校重点领域专项（2022ZDZX4047）；韶关学院重点项目（SZ2022KJ05）；国家自然科学基金（31901537）；韶关学院博士启动项目（99000615）；国家级大学生创新创业训练计划项目（202310576009）

详细信息

作者简介:
曾坚（1987 —），男，副教授，主要从事植物基因功能研究，E-mail：zengjian@sgu.edu.cn

通讯作者:
曾力旺（1987 —），女，助理研究员，主要从事植物基因分子生物学研究，E-mail：zengliwang@163.com

中图分类号: S533
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出版历程
- 收稿日期: 2023-07-21
- 修回日期: 2023-10-11
- 网络出版日期: 2023-12-21
- 刊出日期: 2023-12-28

Functions of MeUGT25 in Resistance of Cassava to Bacterial Wilt Disease

1.
Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region/College of Biology and Agriculture, Shaoguan University, Shaoguan, Guangdong　512005, China
2.
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, Hainan　571101, China
3.
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, Guangdong　510642, China

摘要

摘要: 目的克隆木薯中UDP依赖型糖基转移酶（UDP-glycosyltransferases, UGTs）基因MeUGT25，并进行抗枯萎病功能研究，为木薯抗病分子育种提供新的基因资源。方法通过RT-PCR技术从木薯叶片（SC124）中克隆MeUGT25基因。随后，利用病毒诱导的基因沉默（virus induced gene silencing, VIGS）和地毯草黄单胞菌（Xamthomonas axonopodis pv. Manihotis, Xam）侵染试验研究MeUGT25基因在木薯中的抗病功能。结果病菌Xam能显著诱导MeUGT25基因的表达。在3株阳性干扰植株中，qRT-PCR检测显示它们的MeUGT25基因表达量分别降低71%、70%和69%。Xam侵染试验结果表明，叶片接种Xam 6 d后，MeUGT25V-2和MeUGT25V-3植株叶片上的细菌数量相比对照明显增多，但MeUGT25V-1阳性植株的细菌统计数量和对照叶片相比没有显著差异。然而，从叶片的发病情况来看，3个干扰植株叶片上的菌斑均比对照明显。结论降低木薯叶片中MeUGT25基因的表达量会影响叶片抵抗Xam病菌侵染的能力，推测MeUGT25基因在木薯抗枯萎病中发挥正调控作用。
- 木薯 /
- MeUGT /
- 生物胁迫 /
- VIGS /
- 地毯草黄单胞菌
Abstract: Objective Disease resistance to Xamthomonas axonopodis pv. Manihotis (Xam) of cassava related to MeUGT25, a UDP-glycosyltransferases (UGT) gene, was studied for breeding purposes. Method MeUGT25 was cloned from cassava leaves (SC124) by RT-PCR. Subsequently, virus-induced gene silencing (VIGS) andXam infection challenge experiment were conducted to confirm the disease resistance of the plant. Result The expression of MeUGT25 was significantly induced by the presence of Xam. In 3 transgenic plants, qRT-PCR showed reductions in MeUGT25 expression by 71%, 70%, and 69%. In 6 d after an Xam−inoculation, the bacterial counts on the leaves of MeUGT25V-2 and MeUGT25V-3 plants increased significantly, but not of MeUGT25V-1. On the other hand, apparent plaques appeared on the leaves of the MeUGT25 gene silencing plants indicating the lowered MeUGT25 expression had significantly reduced the resistance of cassava to Xam infection. Conclusion Reduction of MeUGT25 expression in cassava mitigated the ability of the leaves to resist invasion by Xam suggesting a positive regulatory role of the gene played in the disease resistance.
- Cassava /
- MeUGT /
- biotic stress /
- VIGS /
- Xamthomonas axonopodis pv. Manihotis

HTML全文

图 1 MeUGT25基因在Xam处理下的表达分析

*表示与0 d差异显著（P＜0.05）。

Figure 1. Expression of MeUGT25 under Xam treatment

* indicates significant difference from control (0 d) (P＜0.05).

下载: 全尺寸图片幻灯片

图 2 pTRV2-MeUGT25干扰载体构建

A：干扰片段克隆；B：pTRV2载体酶切；C：pTRV2-MeUGT25双酶切。

Figure 2. Construction of pTRV2-MeUGT25 vector

A: Cloned fragment of MeUGT25; B: digested pTRV2 vector; C: verified pTRV2-MeUGT25 vector by digestion.

下载: 全尺寸图片幻灯片

图 3 叶片中MeUGT25基因被沉默后的表达水平

*表示与对照（TRV）差异显著（P＜0.05）。TRV: 对照植株；MeUGT25V-1、MeUGT25V-2、MeUGT25V-3：不同干扰植株。

Figure 3. Expression of MeUGT25 after VIGS

* indicates significant difference from control (TRV) (P＜0.05); TRV: control plant; MeUGT25V-1, MeUGT25V-2, and MeUGT25V-3: different silenced plants.

下载: 全尺寸图片幻灯片

图 4 MeUGT25基因被沉默后发病叶片中细菌数量的变化情况

不同小写字母表示处理间差异显著（P＜0.05）。

Figure 4. Bacteria counts in cassava leaves transformed with pTRV-MeUGT25

Different lowercase letters indicate significant difference among treatments (P＜0.05).

下载: 全尺寸图片幻灯片

图 5 MeUGT25基因被沉默后接种细菌的表型

Figure 5. Symptoms on Xam-infected cassava leaves

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Sequences of primers applied

引物名称 Primer	上游引物 Forward Primer（5′-3′）	下游引物 Reverse Primer（5′-3′）	用途 Usage
MeUGT25	gtgagtaaggttaccgaattcTTTGATTGCCCAGATCGTCG	gagacgcgtgagctcggtaccCAGGCTGGTGGCTACAACGG	载体构建
qMeUGT25	CCGGAATTCTTTGATTGCCCAGATCGTCG	CGGGGTACCCAGGCTGGTGGCTACAACG	荧光定量
MeEF1a	TGAACCACCCTGGTCAGATTGGAA	AACTTGGGCTCCTTCTCAAGCTCT	荧光定量
划线部分为上游同源臂/下游同源臂+酶切位点。 Underline shows upstream homology arm/downstream homologous arm with restriction enzyme digestion sites.

下载: 导出CSV

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