Improvement Of Jingxi Fragrant Glutinous Plant Height Variety By Use Of CRISPR-Cas9 Gene Editing System

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.