Effect Of Directional Editing Of Isoamylase Gene ISA1 On Rice Quality

Rice is one of the most important food crops in the world.The energy and nutrients of rice are stored in the endosperm.The development of the endosperm directly affects the synthesis and accumulation of various substances.It is a process in which multiple enzymes are tightly and perfectly coordinated.At present,fine control pathways for its developmental processes is not very clear.Therefore,it has important appcicable value to accelerate the study of endosperm development.In this study,the isoform amylase 1 was site-directed by CRISPR/Cas9 technology to obtain the T₀homozygous mutants and the homozygous T₁ mutants with stable phenotype were studied.The results were as follows:1.Obtaining T₀ transgenic mutant plants.A suitable target site was designed on the ISA1 gene,and the constructed VK005 vector was transformed into Agrobacterium by heat shock.Then Agrobacterium was introduced into the callus of Zhonghua 11 to obtain T₀ transgenic seedlings.Seven mutations were identified by PCR of Hyg and sequencing analysis.We selected phenotypically stable T₁ homozygous mutants cr-isa1-1?type1,single base insertion?and cr-isa1-2?type3,single base deletion?as study objects.2.The agronomic traits of the T₁ mutant have changed.Compared with the wild type,the two mutants showed wrinkling in grain,the grain length was increased by 6.1%and 5.8%,respectively;The grain thickness was decreased by 13.3%and 15.4%,respectively;And the 1000-grain weight was decreased by 43.6%and 43.0%,respectively.The plant height of cr-isa1-1 was reduced by 6.0%and the seed setting rate was reduced by 4%,but there was no significant change in the total grain number per panicle.3.The T₁ mutant amyloplast and starch granule have changes.The cross-section of the iodine-stained mature grain,with lighter staining of the two mutants,indicates less starch accumulation in the endosperm.Further studies showed that the amyloplasts in the mutants did not enlarge and their structures were abnormal,forming transparent spheroids;starch particles could not be observed by scanning electron microscopy.These results suggest that the ISA1 might plays a key role in the initial stage of starch synthesis.4.Physiochemical indexes of T₁ mutant endosperm have altered.The total starch and amylose content of the two mutants was significantly reduced,the total starch was reduced by 34.4%and 40.0%,the wild type amylose content was 14.2%,and the two mutants were almost 0;The contents of the total soluble sugar were 4.3 and 4.5 times that of the wild type,respectively;the amylopectin synthesis was abnormal,the chain of DP?9 was increased,and the chain of DP between 10 and 60 was decreased;the gel consistency of both mutants were increased by 12.6%.The gelatinization temperature of mutants was also significantly reduced.5.The expression levels of starch synthesis-related genes in T₁ mutant have changed.According to RT-PCR,the expression levels of AGP genes AGPL1 and AGPS1 in the cr-isa1-1 were reduced by76.7%and 60.5%,respectively;And the starch synthase genes SSI,SSIIa,SSIIIa and SSIVb were reduced by approximately 35.9%,33.6%,82.0%and 18.3%,respectively;The starch branching enzyme genes BE1 and BEIIb were decreased by 63.1%and 65.2%,respectively;The isoamylase genes ISA1,ISA2 and PUL were decreased by 60.8%,13.9%and 75.8%,respectively.The expression level of gene PHOL was reduced by 60.8%;However,the expression levels of AGPL2 and ISA3 were 1.26-fold and2.6-fold higher than those of wild-type.In the mutant cr-isa1-2,the relative expression level of the above genes was basically consistent with that of cr-isa1-1.Thus,above results suggested that ISA1affects not only the synthesis of starch but also the expression of other genes...