【作者】 王新；

【导师】 李容柏；

【作者基本信息】 广西大学 ， 作物遗传育种学， 2018， 硕士

【摘要】 传统优质水稻具有符合大众需求的优良品质,同时存在影响其产量的性状。因此,优质水稻的改良和新品种的培育成为我国水稻育种的关键。基因编辑技术的兴起无疑为科学界掀起了一股热潮,目前应用最为广泛的当属CRISPR/cas9基因编辑技术。随着CRISPR/Cas9技术的发展成熟,运用基因编辑技术对水稻基因定点编辑,从而获得性状极优的品种,已成为我国水稻育种的热点。靖西香糯是广西著名的地方水稻品种,米饭水晶状、糯性强、香味浓、食味佳,是优质糯稻的极品,同时存在高杆易倒伏、产量低的缺点,改良其株高特性同时又保持品种的传统风味是本研究要实现的目标。本研究运用CRISPR/Cas9技术定向编辑调控靖西香糯株高的OsGA20ox2(SD1)基因,得到矮化的OsGA20ox2基因突变株,选育出无外源基因的半矮秆纯合突变株系,使该糯稻的植株形态和产量潜力得到显著改良,主要研究结果如下:1.通过国家水稻数据中心获得OsGA20ox2基因的全序列,根据该序列利用生物信息软件MEGA6进行序列分析,同时通过在线软件NCBI在OsGA20ox2基因的主要功能结构域内进行进行引物的设计,利用设计好的引物扩增靖西香糯亲本的功能结构域并送生物公司测序,获得该基因的功能结构域序列。同时运用MEGA6进行亲本材料测序序列与已报道基因序列的差异性分析,确定序列的准确性。根据所测序的靖西香糯序列利用CRISPR-P在线工具设计两个高效靶点,构建具有潮霉素抗性的双靶点OsGA20ox2基因编辑载体,载体构建完成通过酶切和测序的方式对所构建载体进行验证,验证无误后对靖西香糯进行农杆菌介导转化,通过转化共获得了 28株靖西香糯转基因水稻;2.分别对T0靖西香糯进行靶位点PCR扩增及测序分析,分析所有转化材料的突变情况。经检测分析T0靖西香糯转化植株突变率为85.7%,其中2个靶点同时突变频率占总突变的58.3%,双靶点纯合突变高达42.8%,其中3株(12.5%)是T-DNA-free纯合突变植株;3.对获得的T0代水稻植株叶片进行内源GA含量的测定,本研究测定采用水稻幼苗叶鞘生长“点滴法”的生物鉴定方法,利用在一定范围内叶鞘的伸长与赤霉素的浓度成正比的原理,是最常用对的生物鉴定方法之一,具有耗时短,所需样品少,灵敏度高的优点。经鉴定半矮秆突变株材料的内源GA水平显著降低,验证了株高性状受内源GA影响的结果;4.通过对T0和T1代农艺性状的考察,表明与野生型原亲本相比,部分性状如穗数、穗长、剑叶长、每穗粒数和单株产量均有明显得差异,即半矮秆突变株系穗数增加、穗长略缩短、剑叶长变短、穗粒数略减少、单株产量增加,而剑叶宽、结实率、千粒重等性状没有显著差异,表现为显著的矮秆效应。米质方面,粒型无明显差异,糯性和香味无变化,直链淀粉含量为1.2%,与野生型相同。半矮秆突变株系株高由亲本的145.4cm降到113.1cm,降幅32.3cm或22.2%。本研究为培育矮秆香糯水稻品系提供了帮助,表明CRISPR/Cas9应用可高效地定向改良特异的传统水稻品种,为传统水稻品种创新利用提供了有效的技术手段,本研究结果为传统资源利用提供了一个范例。更多还原

【Abstract】 Traditional high-quality rice has good quality that meets the needs of the public,and it also has traits that affect its yield.Therefore,the improvement of high-quality rice and the cultivation of new varieties have become the key to rice breeding in China.The rise of gene editing technology undoubtedly set off an upsurge for the scientific community.Currently,the most widely used technology is the CRISPR/cas9 gene editing technology.With the development of the CRISPR/Cas9 technology,the use of gene editing technology for the fixed-point editing of rice genes to obtain highly traitable varieties has become a hot spot for rice breeding in China.Jingxi are well-known local rice varieties in Guangxi.The rice is crystal-like,strong and strong in flavor,and has a good taste.It is the best quality Japonica rice.At the same time,it has the disadvantages of easy lodging of tall stems and low yield.Its plant height characteristics while maintaining the traditional flavor of the variety is the goal to be achieved in this study.In this study,the OsGA20ox2(SD1)gene of the plant height of Jingxi camphor was directionally edited using CRISPR/Cas9 technology to obtain a dwarf OsGA20ox2 gene mutant and a semi-dwarf homozygous mutant strain with no foreign gene was selected.The indica rice plant morphology and yield potential have been significantly improved.The main findings are as follows:1.The full sequence of the OsGA20ox2 gene was obtained by the National Rice Data Center.Sequence analysis was performed using the bioinformatics software MEGA6.The primers were designed in the main functional domain of the OsGA20ox2 gene using the on-line software NCBI and amplified using the designed primers.The functional domain of the parent of the Jingxi camphor was sent to the company for sequencing,and the functional domain sequence of the gene was obtained.Simultaneously,MEGA6 was used to analyze the difference between the parental material sequencing sequence and the reported gene sequence to determine the accuracy of the sequence.According to the sequencing of the Jingxi camphor sequences,two highly efficient targets were designed using the CRISPR-P online tool to construct a double-targeted OsGA20ox2 gene editing vector with hygromycin resistance.The vector construction was completed by enzyme digestion and sequencing.The vector was constructed and verified.After confirmation,the Agrobacterium tumefaciens-mediated transformation of Jingxi.A total of 28 transgenic rice plants were obtained through transformation.2.The target site PCR amplification and sequencing analysis wereperformed on T0,respectively,and the mutations of all transformed materials were analyzed.The mutation rate of T0 transformed plants was 85.7%,of which 2 target mutations accounted for 58.3%of the total mutations at the same time,and the homozygous mutation at the double target was 42.8%,of which 3 strains(12.5%)were T-DNA-free homozygous mutant plants.3.The endogenous GA content of the leaves of the T0 generation rice plants obtained was determined.In this study,a bioassay method using the "droplet method" for the growth of rice seedling sheaths was used.The elongation of leaf sheaths was proportional to the concentration of gibberellin in a certain range.The principle is one of the most commonly used bioassay methods.It has the advantages of short time,fewer samples,and high sensitivity.The endogenous GA levels of the semi-dwarf mutants were significantly reduced,confirming the results of the effects of endogenous GA on plant height traits.4.By examining the agronomic traits of the T0 and T1 generations,it was shown that some of the traits,such as panicle number,panicle length,flag leaf length,number of grains per panicle,and yield per plant,were significantly different from those of the wild type parent.Dwarf mutants had increased panicle number,slightly shorter panicle length,shorter flag leaf length,slightly reduced number of panicles,and increased yield per plant,but there were no significant differences in flag leaf width,seed setting rate,and 1000-grain weight,showing significant differences.Dwarf effect.In terms of rice quality,there was no significant difference in grain type,and there was no change in sputum and aroma.The amylose content was 1.2%,which was the same as wild type.The plant height of the semi-dwarf mutant decreased from 145.4 cm to 113.1 cm,a decrease of 32.3 cm or 22.2%.This study has provided assistance for the cultivation of dwarf taro rice lines,showing that the CRISPR/Cas9 application can efficiently direct and improve specific traditional rice varieties and provide effective technical means for the innovative utilization of traditional rice varieties.The results of this study provide an example for the use of traditional resources.更多还原