Research On Rice Aroma And Waxy Traits Improvement Based On CRISPR/Cas9 Gene Editing Technology

The demand of consumer for rice has changed from“eating full”to“eating well”with the economic and social development and the remarkable improvement of people’s living standards.The direction of rice genetic and breeding improvement also changed from"paying attention to high yield"to"paying equal attention to high quality and high yield".Recently,CRISPR/Cas9,as a new molecular biology method for target site specific gene editing,has been widely used for rice genetics improvement.In this study,we used CRISPR/Cas9 to edit the rice Waxy（Wx）and BADH2,thus improving the waxy and aromatic traits of rice,respectively.Then the grain quality and agronomic traits of gene editing stable lines were investigated and analyzed in detail.The main research and results are as follow.（1）The rice varieties Ningjing1（NJ1）and Huazhan（HZ）were selected to be knocked out the Wx gene,and the genotype background detection showed that both of them were Wx^ballele.The rice varieties Ningjing1（NJ1）、Huanghuazhan（HHZ）and the CMS line Taonong1A were chosen to be knocked out the BADH2 gene,and the results showed that NJ1 and HHZ were non-fragrant rice,Taonong 1A is an aromatic sterile line and its BADH2 gene has5bp deletion and 3bp mutation in exon 8.（2）We used the VK005-1 vector to construct the CRISPR/Cas9 gene editing expression vectors of the two target genes,and successfully obtained the gene editing homozygous lines by genetic transformation.（3）We identified the phenotype of the homozygous gene editing lines.The mature seeds of Wx knockout lines were stained with 0.02%I₂-KI,and they were dyed purplish red,while the wild type seeds were dyed blue-black.Moreover,scanning electron microscopy of natural cross-sections of mature seeds found that the wild-type starch granules had regular and sharp edges compared to the Wx gene knockout lines.The leaves of BADH2 knockout lines were soaked with 1.7%KOH to identify rice aromas,and all gene-edited homozygous lines can produce fragrance,while the wild type has no fragrance.（4）We adopt the method of PCR and hygromycin（50 mg/L）to screen out homozygous lines of each editor which have transgenic components removed.At the same time,potential off-target sites were detected for Wx and BADH2 knockout plants.（5）We detected the rice quality of homozygous lines with no transgenic components of the Wx knockout.We found that the amylose content of the Wx knockout lines were significantly reduced compared to that of wild-type,and there was no significant change in the total starch content.The starch chain length distribution has changed,and the gel consistency has increased significantly.The RVA spectrum analysis showed that the Wx knockout lines reached the peak viscosity（PV）faster than wild-type,and then quickly decreased and maintained to a low level of viscosity.The rice flour urea swelling experiment showed that the swelling characteristics of rice flour changed significantly.（6）The measurement of fragrance 2-AP on homozygous lines with T-DNA-free of the knockout BADH2 showed that the content of 2-AP in the BADH2 knockout lines was significantly higher than that of the wild type which could not be detected.Rice grain quality measurement revealed that there was no significant change in amylose content,gel consistency,RVA profile,and rice flour swelling characteristics.The gelatinization temperature（GT）showed that the GT of nj1-cr^BADH2-1 and hhz-cr^BADH2-1were significantly lower than that of wild type,while other knockout lines did not change significantly.（7）We used the homozygous line of the non-transgenic component of the knockout BADH2 in Huanghuazhan genetic background to cross with Taonong 1A（B-TYXZ）to improve the aroma characteristics of Taoyouxiangzhan（TYXZ）varieties.The aroma substances measurement showed that the 2-AP content of B-TYXZ was significantly higher than that of wild type TYXZ.Compared with wild-type TYXZ,the amylose content and gelatinization temperature of B-TYXZ did not change significantly,while the gel consistency and the adhesiveness significantly increased in B-TYXZ,and the fresh rice hardness and the regenerated rice hardness decreased significantly in B-TYXZ.The food taste measurement showed that the grain appearance score and the taste score decreased significantly in B-TYXZ,but the comprehensive score of B-TYXZ did not change significantly.（8）The determination ofγ-aminobutyric acid（GABA）content in the improved fragrance of Taoyouxiangzhan（B-TYXZ）and wild type Taoyouxiangzhan（TYXZ）found that the content of GABA in B-TYXZ was significantly lower than that in TYXZ,...