MiRNAs And Their Targets Related To Tuber Root Development And Starch Accumulation In Cassava

MicroRNAs (miRNAs) are a group of non-coding small RNAs with usually21-24nt in size which of mediated target genes involved in almost all essential biological processes throughout transcription, post-transcription and translation levels. Cassava is the most important lubery cropin the tropics supplied staple food for600million people in the world. It is known as much higher starch yielding and tolerance to drought and barren soil conditions. It will be a new strategy of molecular breeding try to find miRNAs played a key role in tuber development and starch accumulation in cassava. In the study, cultivar Arg7with high tuber root starch content and wild species W14with low starch content were used to construct three miRNA libraries:Arg7leave, Arg7tuber roots and W14tuber roots. The Solexa deep sequencing and bioinformatics analysis based on genome sequences were used to indentify miRNAs and their targets related to tuber root development and starch accumulation. Part of miRNAs and targets have been verified through real time fluorescence quantitative PCR (Q-PCR). The main results shown as the followings:(1) Through Solexa deep sequencing technology and bioinformatics analysis,167miRNAs were found from the three small RNA libraries.The93conserved miRNAs can be attributed to21miRNA families, and the other74miRNAswere newly discovered.167miRNAs reads were normalized and took more than1000reads as high expression of miRNA. The81miRNAs in Arg7leaf are high expressed, high expression in the Arg7root are57, and high expressionin the root tuber of W14are58. The48miRNAs are highly expressed in the three libraries. In addition, the miRNAs high expression in the high-starch varieties Arg7and expression level in root tuber is higher than that in the root leaves are novel₄2, miR394abc, novel₅2, novel₂0, miR319bcdef and miR164bcd.(2) Through Hitsensor software to predict167miRNA target genes, the complementary sequences of all miRNA molecules were searched from the U.S. Department of Energy JGI cassava CDS sequence database. The93conserved miRNAs corresponding to146target genes and115target genes relate to74new miRNAs. These target genes functioncan be divided into four categories:metabolism proteins, transcription factors, signal transduction proteins and stress proteins. Target genes (the corresponding miRNAs) associated with sugar metabolism and starch synthesis are SPS2F (miR394abc), AGPase (miR394abc),3-ketoacyl-CoA synthase11(novel-52), starch synthase (miR159) and UDP-glycosyltransferase (miR156) respectively.(3) Eight three miRNAs were identified through Multiplexed RT method in leave and tuber roots of cassava. There were6miRNAs, miR172d,miR396c,miR398a,miR398b, miR399e and miR399f were only highly expressed in tuber roots of Arg7, other3miRNAs, miR397, miR169defg and novel-42were highly expressed in fiberous roots of Arg7, and13miRNAs, miR160b, novel-10, novel-19, novel-20, novel-22, novel-50, novel-51, novel-6ab, nevel-43abcd, novel-31abcd, miR164abcd, novel-55-3p highly expressed in leave of cassava.(4) Using real time PCR technique to relative quantitative analysis of the28miRNAs, the results indicated that miR169abcd and novel-52are highly expressed at the seedling stage; the miRNAs (novel-2, novel-20, novel-50, novel-55-3p, miR398a, miR399e and miR399f) are high expressed in expansion period; the miR160b, miR394abc, novel-6ab, novel-10, novel-19, novel-31abcd, novel-43ab, novel-51and novel-42are highly expressed in maturation stage. Furthermore, the results of Relative quantitative analysis are more consistent with the sequencing results.(5) Made a synthetically study of sequencing, relative quantitative analysis and target genes prediction results, the two target gene SPS2F and AGPaseof miR394were selected to quantitative analysis, and compare the two target genes'results were regulated by miR394. In the root tuber maturity stage, which miR394have a high expression, the two targets genes are lowly expressed. Using SAS software to do linear regression correlation analysis two target genes with miR394. Correlation coefficients (-0.9975and-0.5350) were calculated respectively. Both of the two targets genes were negatively related by miR394. Taken together, miR394negatively regulates the two target genes then control the development of root tuber. All the results lay the foundationsfor further elucidating the regulation mechanism by miR394in cassava.