Salt Tolerance And Yield Improvement Of Japonica Rice Based On CRISPR/Cas9 System

Rice?Oryza sativa L.?is one of the most important food crops in the world.Feeds more than half of the world's population.Salt stress is an important environmental factor that restricts the growth and development of rice.High concentration of salt stress at seedling stage will directly inhibit the growth and water use of rice,resulting in a significant reduction in rice yield.In recent years,the total area of saline soil in China is showing a trend of gradually expanding.Therefore,improving the salt tolerance of rice and increasing rice yield is of great significance to China's food security.Os DST,Os EIL1 and Os EIL2 are the transcription factors in rice.Previous studies have shown that these three gene mutations will lead to increased salt tolerance in rice,In addition,the Os DST gene mutation will also increase rice yield.CRISPR/Cas9 system is widely used in rice genetic improvement because it can induce rapid site-directed mutations in the rice genome.In this study,a japonica rice variety Dongnong427 was used as material and two transformation vectors were constructed to knock out Os DST,Os EIL1 and Os EIL2 genes respectively by CRISPR/Cas9system.The mutants with salt tolerance and yield increased were successfully obtained through agrobacterium mediated genetic transformation,transgenic detection and mutation detection methods.The specific research contents and results are as follows:1.Construct CRISPR/Cas9-DST vector to knocked out Os DST gene and CRISPR/Cas9-EIL1-2 vector to knocked out Os EIL1 and Os EIL2 gene.Two vectors were transformed into dongnong427by agrobacterium mediated method.26 and 25 positive T₀ plants were obtained respectively.2.Specific primers were designed to perform PCR amplification and sequencing the positive plants transformed with two vectors.21 Os DST mutants were obtained,1 of them was homozygous mutant?4.8%?,6 were heterozygous mutants?28.6%?,13 were bi-allelic mutants?61.9%?,and 1was chimeric mutant?4.8%?.5 Os EIL1 and Os EIL2 mutants were obtained,4 of them were heterozygous mutants?80%?and 1 was bi-allelic mutant?20%?.Off-target analysis results demonstrate that the 6 potential off-target sites of 21 Os DST mutants and 5 potential off-target sites of 5 Os EIL1 and Os EIL2 mutants had no off-target events.3.In T₁ generation,dst-25,dst-17 and eil1/2-9 were seeded into lines.The homozygous mutants were screened by using specific primers.Hyg-F/R and Cas9-F/R primers were used to screen the mutants without T-DNA element insertion.Three kinds of Os DST mutants without T-DNA element insertion were obtained,including 12,6 and 5 plants respectively,and one kind of Os EIL1 and Os EIL2 mutant without T-DNA element insertion,including 4 plants.These plants were propagated to T₂ generation,named dst HM1,dst HM2,dst HM3 and eil1-2HM respectively.4.In the T₂ generation,mutants were cultured to the seedling stage by hydroponics.The seedlings cultured to 12 days old were treated with 200 m M Na Cl solution for 4 days.After treatment,the growth of all mutants were significantly better than wild type,the reduction of fresh weight and dry weight,the Na⁺/K⁺of shoots and roots were significantly lower than that of wild type.After 5 days recovery,most wild type were wilted and died,while the mutants still had a higher survival rate.The survival rates of dst HM1,dst HM2 and dst HM3 were 84.33%,81.33%and 85.00%respectively,the survival rate of it's wild type control group was 7.0%.The survival rate of eil1-2HM was 65.06%and the survival rate of it's wild type control group was 3.03%.5.The yield characters of mutants were investigated at maturity.The number of effective panicles,primary branches,secondary branches and yield per plant of the three dst HM mutants were all significantly higher than that of the wild type.And there were no significant difference between the yield characters of eil1-2HM mutant and the wild type.These results showed that mutation of Os DST gene could also improve the yield of rice,mutation of Os EIL1 and Os EIL2 gene didn't affect the yield of rice.