| Inositolphosphorylceramide synthase（IPCS）is one of the key enzymes in the process of sphingolipid metabolism and plays an important role in the biosynthesis of sphingolipids.It was widely found in yeast,protists,plants and other organisms.Studies have found that phytosphingolipids and their metabolites are involved in the regulation of many important life process,such as cell growth,differentiation,senescence and programmed death.The rice（Oryza sativa L.）is an important food crop.There are three OsIPCS genes in its genome:OsIPCS1,OsIPCS2 and OsIPCS3.However,there are few reports about the function of OsIPCS family genes of rice.In this study,we constructed the CRISPR/Cas9 knock out vectors of OsIPCS family genes,and were transformed into callus of Oryza sativa L.subsp.japonica zhonghua 11 by Agrobacterium mediated method.The T0 generation differentiation seedlings were obtained by means of plant tissue culture technology.The T0 plants were identified by molecular detection and sequencing,and transgenic plants with different mutation types were obtained.Quantitative PCR（qRT-PCR）technology was used to detect the transcription level of corresponding genes of various types of mutants in the normal planting environment,and the predicted amino acids were coded based on the mutated loci.Under the normal planting conditions in the experimental field,phenotypic and corresponding indicators were observed during rice growth and development in single gene mutations and multiple gene mutations（dual-gene and three-gene mutations）,initially revealing the function of OsIPCS family genes on growth and development of rice.It lays a foundation for the molecular breeding of rice and has important theoretical or practical significance.The main research results are as follows:1.Successfully constructed four vectors that pYLCRISPR/Cas9-OsIPCS1,pYLCRISPR/Cas9-OsIPCS2,pYLCRISPR/Cas9-OsIPCS3 single gene knock out vectors with one target site and pYLCRISPR/Cas9-OsIPCS1/2/3 three genes knock out vector.Then those vectors transformated in zhonghua 11 callus respectively by Agrobacterium mediated method,and differentiated T0 plants through plant tissue culture technology.The PCR amplification of glufosinate（bar）gene was performed on T0 transgenic plants.The results showed that the transformed OsIPCS single gene knock out vectors obtained 33,30,and 50 positive transgenic plants,respectively;the transformation of three knock out vectors yielded 134 positive transgenic plants.2.Sequencing the knock out target sites of T0 transgenic plants,revealed that CRISPR/Cas9 systems have different editing efficiency for each OsIPCS genes.OsIPCS1 obtained two types of mutation（heterozygous mutation and inserted G base）,the mutation rate is 6%;OsIPCS2 obtained six types of mutation（inserted A base,deleted T base,deleted 4,5,6 bases and heterozygous mutation）,the mutation rate is90%;OsIPCS3 has three types of mutation（inserted T base,deletion 9 bases and heterozygous mutation）,the mutation rate is 18%.Sequencing analysis of the transgenic plants that transformed with the three gene knockout vector,revealed that7.5%of the mutations were simultaneously mutated for the three genes,62%for the OsIPCS1/2 mutation,and 1.5%for the OsIPCS2/3 mutation.No OsIPCS1/3 and OsIPCS3 knockout plants were obtained.The Os IPCS1 and the OsIPCS2 single gene mutation accounted for 20%and 9%of the mutation ratio.3.qPCR was used to detect the transcription level of homozygous mutant gene under normal planting conditions.It can be seen that OsIPCS2 gene transcription levels of different types of osipcs2 mutants were down-regulated to varying degree,and the type of deletion T mutation was significant.However,there was no significant difference on osipcs3 mutant and wild type in the transcription level of OsIPCS3 gene.The amino acid encoded by the mutated gene was predicted and the results showed that frame mutations occurred in all other mutation types except that the deletion bases was an integer multiple of three.4.Phenotypic observation of the OsIPCS family genes mutant strains and the wild type zhonghua 11 in whole growth period under the normal planting conditions.We discovered the osipcs2（-T）mutants plant height is about 76%of wild type,the seed setting rate is 10%lower than the wild type;the osipcs1/2 mutants had only 61%of the wild type plant height,and the seed setting rate was 82%lower than the wild type;the osipcs1/2/3 mutants was impediment on growth and development growth that plants were dwarfed severely,the number of tillers was less and no seeds.5.This research creates a batch of transgenic mutants of rice that stable genetically modified and without T-DNA insertion by CRISPR/Cas9 technology,which valuable genetic material for further study the functions of the OsIPCS family. |
