【作者】 陈林；

【导师】 张泽民；

【作者基本信息】 华南农业大学 ， 作物遗传育种， 2021， 硕士

【摘要】 水稻（Oryza sativa L.）是植物基因组学研究的重要模式作物之一,水稻的产量严重影响着我国的粮食安全问题。水稻叶片形态是重要的农艺性状,对于植株产量起着重要的作用。因此,挖掘水稻叶形态相关基因及探究其分子机制,有利于通过改良株叶形态来提高产量。本研究以一个水稻叶片窄卷曲的突变体rel3为研究材料,通过表型鉴定、细胞学切片、遗传分析、基因定位、干旱处理、实时荧光定量PCR等方法,克隆了一个控制水稻叶片窄卷曲性状的基因REL3及初步探索了其耐旱性,主要研究结果如下:1、从三叶期开始,突变体rel3植株的叶片要明显比野生型窄小且向内卷曲,之后伴随整个生育期;相比于野生型,rel3的叶色加深、单株产量升高、粒长变短、粒宽变宽以及株高变矮;比对rel3和野生型的叶绿素含量发现,rel3在生长发育后期叶片的叶绿素含量增加导致其叶色加深;测量rel3和野生型的茎节长度发现,rel3茎节长度变短导致其植株变矮。2、通过细胞学结构观察发现,在维管束方面,突变体rel3的大维管束数目减少25.75%,小维管束数目减少44.23%,大、小维管束数目减少共同导致了叶片变窄;在泡状细胞方面,突变体rel3泡状细胞数目增多89.52%,泡状细胞面积减少20.45%,泡状细胞数目增多和泡状细胞面积变小共同导致了叶片向内卷曲。3、突变体rel3和平展叶材料R65的杂交后代连续种植,通过对F₁单株及F₂、F₃群体进行遗传分析发现,rel3窄叶和卷叶性状共同遗传,并且受一对隐性基因控制;利用BSA测序和分子标记相结合的方法,将控制窄卷叶性状的基因精细定位在3号染色体RM3280和RM14830区间内,对区间里的基因测序发现,LOC＿Os03g19520基因在突变体rel3的第14个外显子（+35位置）有一个碱基T的缺失,使得移码及翻译提前终止,另外LOC＿Os03g19520在突变体rel3中的表达量比野生型显著性下降,推测LOC＿Os03g19520为REL3的候选基因。4、q RT-PCR分析REL3的表达模式,发现REL3在全生育期都有表达,其中在幼穗中表达水平最高,在根的表达水平最低;与野生型相比,ACL1、ACL2、ADL1、REL2、RL14、ROC5和SRL2等卷叶基因在突变体rel3中表达量下降,REL3可能与这些卷叶基因存在关联。5、干旱处理后,突变体rel3的失水率升高、SOD活性下降、耐旱性下降;与野生型相比,Os DREB1A、Osb HLH148、Os DT11、Os MYB2及Os SAPK2等耐旱基因在突变体rel3的表达量下降,REL3可能下调相关耐旱基因表达量,从而导致了突变体rel3耐旱性的下降。综上所述,本研究克隆了一个水稻的窄卷叶基因REL3,同时初步探索了REL3的耐旱性功能,为以后叶片发育的分子机制研究奠定了重要基础。更多还原

【Abstract】 Rice is one of the significant model crops for plant genomics research,and the output of rice has a serious impact on the country’s food security issues.As an important agronomic trait,rice leaf morphology plays an important role in plant yield.Therefore,it is of great significance to study the mechanism of rice leaf morphology-related genes for breeding superior and high-yield rice varieties.Here,a narrow rolled leaf mutant rel3 was used as the research material.Through phenotypic identification,cytological sectioning,gene localization,drought tolerance test,real-time fluorescent quantitative PCR and other test methods,we cloned the gene REL3 and preliminarily explored the drought tolerance of REL3.Key research findings were shown below:1.Since trefoil stage,the leaves of mutant rel3 are narrower and roller than wild type in the whole growth period;Besides,compared with wild type,rel3 has greener leaf color,higher yield per plant,shorter and wider grain,and displays shorter plant height.Comparing the chlorophyll content of the wild-type,it was found that the chlorophyll content of rel3leaves increased a lot in the later growth and development stage,which was the reason for the deepening of the leaf color of rel3.By measuring the Internode length of rel3 and wild-type,it was found that the shorter Internode length of rel3 resulted in shorter plant height.2.Through cytological structure observations,it is found that the number of large vascular bundles of rel3 decreased by 25.75%,and the number of small vascular bundles decreased by 44.23%.So,the reduction in the vascular bundles’number of rel3 causes the narrowing of leaves.On the other hand,the number of vesicle cells in rel3 increased by89.52%,and the area of vesicle cells decreased by 20.45%.The increase of vesicle cells’number and the decrease of the vesicle cells’area results in the curling inward of rel3’s leaves.3.The genetic analysis of hybridization including F₁ individual plant,F₂ population,F₃population of mutant rel3 and flat leaf material R65 indicates that the narrow leaf and curled leaf of rel3 are inherited together and controlled by a pair of recessive genes.Through BSA sequencing and Molecular Mapping,the gene was located between RM3280 and RM14830.Then,by sequencing the gene in this region,it is found that in rel3,there is a single base deletion in the 14th exon of LOC＿Os03g19520,which leads to premature translation termination.Therefore,LOC＿Os03g19520 was identified as a candidate gene for REL3.4.The expression pattern of REL3 was analyzed by q RT-PCR,and we found that REL3was expressed throughout the growth period,the expression level of REL3 was the highest in young spike and the lowest in root.Referring to the expression level of the wild type,the expression of leaf rolling genes such as ACL1,ACL2,ADL1,REL2,RL14,ROC5 and SRL2was down-regulated,indicating that REL3 may be associated with these leaf rolling genes.5.After drought stress treatment,the water loss rate of rel3 increased,and the SOD activity decreased,which indicates that rel3’s drought tolerance decreased.What’s more,the expression of drought tolerance genes such as Os DREB1A,Osb HLH148,Os DT11,Os MYB2,Os SAPK2 decreased.So REL3 may down-regulate the expression of related drought-tolerant genes,resulting in a decrease in the drought tolerance of rel3.Overall,we cloned a narrow curled leaf gene REL3,and initially explored the drought tolerance function of REL3,laying an important foundation for the future study of the molecular mechanism of leaf development.更多还原