Profiling Mirnas And Their Target Genes Involved In Citrus Fruit Development

Citrus is one of the most important fruit. To improve the quality of citrus fruit has always been one of the most vital goals of citrus breeding work. Studies on molecular mechanism that regulates citrus fruit development will provide a solid theoretical basis for molecular breeding of citrus. Besides well-known transcription factors(TFs), microRNAs(miRNAs) widely involved in different stages of plant development are also a vital class of regulation factors during fruit development. miRNAs, typically 20-24 nucleotides(nt) in length, are generated from MIR genes that contain a stem-loop secondary structure. After processed into mature miRNAs, they regulate gene expression mainly at the post-transcriptional level. Here, a high-throughput sequencing approach is used to characterize miRNAs and their target transcripts in citrus. Consequently, we identify key miRNAs and their target genes(miR164-Cs5g10870(NAC TF) and miR477a-3p-Cs6g19680(SEP TF)) which are involved in citrus fruit development from sequencing data. Then we analyze the biological functions of these key mi RNAs and their target genes during fruit development and ripening. Additionally, potential TAS genes and phasiRNAs in sweet orange are identified through analyzing sRNAome and degradome data. Furthermore, we analyze the function of miR3954 a and phasiRNAs derived from Cs1g09600 and Cs1g09635 targeted by miR3954 a. Detail results are showed as follow:1. Identify citrus miRNAs and their target genes through sRNAome and degradomeWe use a high-throughput sequencing approach to characterize the miRNAs and their target transcripts in leaf, flower and fruit of sweet orange(Citrus sinensis), flavedo and pulp of pummelo(C. grandis). The genome sequences of sweet orange(data published) and pummelo(data unpublished) are used as reference sequences. Consequently, we identify 183 known miRNAs and 38 novel miRNAs in sweet orange; 106 known miRNAs and 12 novel miRNAs in pummelo. In addition, 405 targets are identified for 107 miRNAs in sweet orange leaf; 265 targets are identified for 166 miRNAs in sweet orange flower; 322 targets are identified for 118 mi RNAs in sweet orange fruit.2. Identification and function analysis of mi RNAs and their target genes involved in citrus fruit development and ripeningBased on the data analysis of sRNAome in sweet orange leaf, flower and fruit, we obtain 80 miRNAs which are higher expressed in sweet orange leaf or flower or fruit. Then the sequencing data are further confirmed by Quantitative RT-PCR(qRT-PCR) and RNA gel blotting analyses to select the remarkable higher expressed mi RNAs in leaf or flower or fruit. As a result, miR482d-5p.2, miRN03 and mi RN36 are higher expressed in sweet orange leaf; miR3954 a, miR167 b.2, miR5179 and miRN08 are higher expressed in sweet orange flower; miR3951, miR164, miRN31 and miR477a-3p are higher expressed in sweet orange fruit. Subsequently, 5’RACE and transient expression system in tobacco are used to verify the target genes of mi RNAs. Expression pattern of key miRNAs and their target genes are then further investigated on 8 stages of fruit development and ripening. The expression level of these key miRNAs and their target genes displays a complementary pattern. miR164 shows significantly higher expression level at the final stage(ripening stage), with a steady increase in expression level during fruit ripening. Thus miR164 may have particular function at the fruit ripening stages. miR477a-3p and miRN31 have similar expression patterns. Their expression levels dramatically increase at 140 DAF and drop at 170 DAF stage(color break stage) which means that they may play as a regulator in sweet orange color break stage. To investigate the function of mi R164 and miR477a-3p in citrus fruit development and ripening, over-expression vectors of miR164 and miR164 short tandem target mimic(STTM) are constructed and then transformed into precocious trifoliate orange. Consequently, we obtain 1 positive transformant of OX-miR164 and 4 positive transformants of OX-miR164-STTM. Similarly, over-expression vectors of miR477a-3p and miR477a-3p-STTM are constructed and transformed into Hongkong kumquat. As a result, we obtain 5 positive transformants of OX-miR477a-3p and 3 positive transformants of OX-miR477a-3p-mimic. Due to the long period of transformation and regeneration of citrus explant, we haven’t observed any interesting phenotypes which imply the function of miR164 and miR477a-3p.3. Identification of sweet orange phasiRNAs and function analysis of phasiRNAs triggered by miR3954aIn this study, sRNAome and degradome of sweet orange leaf, flower and fruit are used to identify sweet orange phasiRNAs. Accordingly, the seven miRNAs that trigger their targets to generate phasiRNAs are miR167.2、mi R2118.1、miR482a-3p、miR482c、miR827.2、miR3954a and miRN20 respectively. A total of 235 phasiRNAs, including 48 phasiRNAs with high abundance are generated after these seven miRNAs trigger their target genes. These 48 phasiRNAs target 243 genes participating in different biological process of sweet orange development. miR3954 a targets Cs1g09600 and Cs1g09635, and then generate 8 phasiRNAs with high abundance. To know more about the function of miR3954 a and phasiRNAs triggered by miR3954 a during sweet orange development, over-expression vectors of miR3954 a and miR3954a-STTM are constructed and transformed into Hongkong kumquat. In the issue, we obtain 17 positive transformants of OX-miR3954 a and 1 positive transformant of OX-miR3954a-mimic. Intriguingly, three OX-miR3954 a progenies flower significantly earlier. After further analysis, we observe that phasiRNAs triggered by miR3954 a are also higher expressed in OX-miR3954 a transformants. More, 30 genes targeted by these phasiRNAs present down-regulated in OX-miR3954 a transformants. These results imply that miR3954 a, phasiRNAs triggered by miR3954 a and target genes of those phasiRNAs may form a network regulating the flowering of citrus together.