Regulation Of Millet Non-coding RNA Seita.4G158100.1 In Starch Synthesis

Millet is the product of the peeling of the grain and is rich in many human nutrients,including protein,fat,sugar,many vitamins and essential nutrients such as Ca,P and Fe.Its main component is starch,and understanding the mechanism of starch synthesis is beneficial for targeted improvement of millet food quality,which is of great significance for grain quality improvement.Previous studies have shown that three Lnc RNAs（lnc RNA＿2308,lnc RNA＿1267 and lnc RNA＿1631）are involved in the regulation of several grain traits in rice and have significant effects on the changes of starch content in rice.In this study,we compared the homologs of lnc RNA＿1631 in cereals by Phytozome Blast,and Seita.4G158100.1 was found to encode a segment of Lnc RNA in the cereal genome.The Stlnc RNA vector for targeted editing of Seita.4G158100.1 gene was designed based on CRISPR/Cas9 technology,T₀ generation editing strains were obtained by Agrobacterium infestation,T₂ generation plants were obtained after addition of generations,and Seita.4G158100.1 pure and mutant materials were screened and identified.We investigated the regulatory role of Lnc RNA gene Seita.4G158100.1 in the starch synthesis pathway by analyzing the key enzyme activities and the spatio-temporal expression differences of their editing genes of Seita.4G158100.1 mutant and control material Ci846,observing the sub-microscopic structure of endosperm starch granules morphology,and measuring the amylose and total starch contents.The main findings of this study are as follows:1.The Seita.4G158100.1 gene was successfully edited by CRISPR/Cas9,and three edited pure line materials were obtained:SiA350-22,SiA354-9,and SiA353-7.The results of sequencing revealed that the mutation of G→A occurred at position 104 and TC→GT occurred at position 117-118 in SiA350-22;SiA354-9 had a nucleotide deletion at position 107-109 and a mutation of CTC→NGG at position 116-118;SiA353-7 had a mutation of G→A at position 104 and a mutation of T→G at position 117.2.The total starch content of the 14 cereal materials varied from 513.9 to 590.97 mg/g,and the amylose content ranged from 36.44 to 84.99 mg/g.The edited material SiA353-7 had the highest total starch content and amylose content,while material 115-247 had the lowest total starch content.The total starch content of SiA350-22 and SiA354-9 were 534.47 mg/g and 521.41 mg/g,which decreased by 3.95%and 6.29%,respectively,compared to the control material Ci846 with 556.42mg/g.The total satrch content of SiA353-7 increased by 6.2%compared to Ci846.3.Scanning electron microscopic observation of the starch granules morphology of the gene edited materials and the control materia Ci846 showed that compared with Ci846,the starch granules morphology of the three edited lines showed regular ellipsoidal shape,smooth surface,uniform size,little change in volume from the control materials,less fragmented structure and tight arrangement.4.The mutation of Seita.4G158100.1 significantly up-regulates the expression of SH1 and increased SUS activity,decreased DBE activity.ADPG pyrophosphorylase（AGPase）,Granule-bound starch synthase（GBSSI）,and Soluble starch synthase（SSS）activities are important for starch accumulation.All three enzyme activities of SiA350-22 and SiA354-9 were reduced compared to Ci846,while SUS,AGPase,GBSSⅠ,SSS,and Starch branching enzyme（SBE）activities were increased in SiA353-7 compared to the control,resulting in SiA353-7 having higher starch content.5.26 DAP is a critical period for starch synthesis in millet,and the activity of GBSSⅠis directly related to the level of amylose content,while the level of SSSⅢand SBEⅡexpression affects starch granule morphology.