小麦凝集素激酶TaLecRK-IV.1基因的作用和由纹枯病诱导的小麦基因发展的分子标记

摘要

小麦纹枯病主要由土传真菌Rhizoctonia cerealis侵染小麦茎基部引起。它已经成为世界范围内小麦生产的限制性因素，在中国更为严重。小麦纹枯病的防治已经成为小麦生产可持续发展的主要问题。培育和种植抗性小麦品种是防治小麦纹枯病最环保、高效的方法。小麦纹枯病抗性遗传机制研究有助于解析小麦抗性相关基因的功能和作用。
　　本研究中，从抗纹枯病的小麦材料CI12633中鉴定了一个L型凝集素受体激酶基因，命名为TaLecRK-Ⅳ1。与感纹枯病小麦温麦6号相比，在CI12633中该基因的表达受纹枯病侵染诱导。序列分析表明，该基因编码含有676个氨基酸残基的L型凝集素受体激酶。利用病毒诱导基因沉默技术(virus-induced gene silencing，VIGS)技术沉默CI12633的TaLecRK-Ⅳ.1后，小麦植株表现矮化的表型，表明该基因可能是小麦矮化的负调控因子。进一步在含有矮化基因Rht-D1b(formerly Rht2)的小麦中分析了该基因的转录，结果表面TaLecRK-Ⅳ.1在矮化小麦中的表达量很低。另外，参与植物生长和发育的激素---赤霉素和生长素诱导TaLecRK-Ⅳ1的转录，而脱落酸和水杨酸对该基因的表达没有影响。以上研究结果暗示TaLecRK-Ⅳ.1参与了小麦发育过程。另外，沉默TaLecRK-Ⅳ.1的小麦植株对纹枯病侵染的抗性反应没有变化，暗示该基因在小麦抗纹枯病反应中并没有主要作用。
　　一个包括114个株系的重组自交系群体(RIL)及其亲本山红麦（抗纹枯病亲本）和温麦6号（感病亲本）在大田进行纹枯病抗性评价。抗性表型分析发现，株系的病情指数是持续变化的，表明山红麦对纹枯病的抗性反应是数量遗传的。在microarray中鉴定的纹枯病抗性相关基因基础上，基于这些基因的序列，开发得到8个分子标记，并在RIL群体中鉴定了每个单株的分子标记基因型。其中，三个分子标记(RGA-left，RGA-right and F3F-TC371509R)与纹枯病抗性位点连锁。然而，在该群体中，所有的QTL的LOD值都没有达到显著，没有检测到任何与纹枯病抗性相关的QTL。以上结果主要是由于山红麦/温麦6的RILs纹枯病病级鉴定不准确，另外，山红麦/温麦6的抗性是由微效QTL所控制。另一种原因可能是分子标记所基于的基因不是山红麦抗病反应的重要基因。在以后的研究中，提高纹枯病鉴定准确性，利用更大的RIL群体，或者更多的分子标记，有助于小麦纹枯病抗性遗传机制的解析，提高分子标记辅助选择效率，加快抗病改良育种进程。

目录

声明

摘要

Abstract

Contents

Chapter 1 Introduction and literature review

1．1．1 History and origin of wheat

1．1．2 Social and economic importance

1．1．3 Wheat cultivation around the world

1．1．4 Influence of plant height on wheat production

1．1．5 Role of phytohormones on plant growth

1．1．6 Factor that favors wheat adaptability

1．1．7 Constraints of wheat production

1．1．8 Wheat sharp eyespot

1．1．9 Employment of RNA-seq to identify putative sharp eyespot resistant genes

1．1．10 Gene introgression from wheat wild relatives

1．1．11 Importance of molecular markers in crop breeding

1．1．12 Molecular marker development

1．1．13 Recombinant inbred line(RIL)populations in breeding research

1．2 Literature review

1．2．1 Sharp eyespot

1．2．2 Plants and disease

1．2．3 Plant detense mechanisms

1．2．4 Signal perception

1．2．5 Genes mediated resistances

1．2．6 HypersensitiVe response(HR)

1．2．7 Bioehemical deterises

1．2．8 Pathogenesis-related proteins

1．3 Purpose of the study

Chapter 2 Silencing of L-type lectin-like receptor kinase gene TaLecRK-IV.1 leads to dwarf phenotype in wheat line CI12633

Introduction

2．1 Materials and Methods

2．1．2 RNA Sequencing-based transcriptome analysis

2．1．3 Methodology

2．1．4 Virus-Induced Gene Silencing

2．1．5 BSMV inoculation

2．1．6 Test for susceptibility to sharp eyespot

2．1．7 Phytohormones treatment(GA，Auxin，ABA and SA)

2．2 Results

2．2．1 Identification of TaLcRK-IV.1 by RNA-Seq and cloning of the gene sequence

2．2．2 TaLcRK encodes an L-type lectin domain containing receptor kinase IV.1

2．2．3 Chromosomal location of TaLecRK-IV.1

2．2．4 Phenotype resulting from the gene silencing experiment

2．2．5 Sharp eyespot responses of the plants subjected to the VIGS experiment

2．2．6 Relative expression of TaLecRK-IV.1 and its homologue gene in wheat line with Rht-D1b

2．2．7 Analysis of the expression of GA biosynthetic enzymes TaGA20ox and TaGA3ox2 in Rht2 wheat line and CI12633 plants inoculated with BSMV:TaLecRK-IV.1

2．2．8 Expression patterns of TaLecRK-IV.1 in response to exogenous hormone treatments

Discussion

Chapter 3 Analysis for quantitative loci using molecular markers derived from wheat genes induced by sharp eyespot

Introduction

3．1 Materials and methods

3．1．1 Plant materials

3．1．2 Pathogen material

3．1．3 Pathogen tests

3．1．4 Pathogen assessment

3．1．5 DNA isolation and PCR amplification

3．1．6 Linkage map construction and QTL analysis

3．2 Results

3．2．1 Phenotypic assessments of sharp eyespot infection

3．2．2 Marker development，genotyping and linkage map construction

3．2．3 Analysis of the phenotypic and genotypic data for QTL associated with the resistance trait

Discussion

Chapter 4 General discussion

Summary

References

Acknowledgements

Funding

Manuscript in preparation