Functionalanalysis Of High Temperature Response LncRNAs Regulating Anther Fertility In Cotton

Cotton is an important economic crop in China due to its fiber(as a main textile raw material)and seed(contains high oil content).In summer,cotton reproductive development often encounters continuous high temperature(HT)weather.HT affect the cotton male reproductive organs development and can lead to male sterility.Long non-coding RNA(lncRNA)plays an important regulatory role in response to adversity stress and multiple processes of biological development.Our previous study have comprehensively identified cotton lncRNAs,of which 4,712 lncRNAs were predominantly expressed in cotton anthers.Further HT-induced expression analysis was performed in ‘84021’ and HT-sensitive line ‘H05’,and finally 25 lncRNAs that were predominantly expressed in anthers and responded to HT,were screened.In this study,we selected 4 lncRNAs from 25 lncRNAs which responded to high temperature at different anther development stages for further researches.The effects of these 4lncRNAs on cotton anther development under HT stress were explored through genetic transformation and phenotyping methods.The research results are as follows:1.4 RNAi vectors of lncRNAs were constructed,and transgenic cotton lines were obtained through genetic transformation.MLinc12 obtained 6 transgenic plants,MLinc15 obtained 72 transgenic plants,MLinc20 obtained 15 transgenic plants,and MLinc22 obtained 18 transgenic plants.2.All T0 generation RNA interference(RNAi)plants of the 4 lncRNAs showed anther abortion.Among them,the MLinc12 RNAi plants showed small flowers,sparse and indehiscent anthers,and few pollen grains in the anthers;the MLinc15 RNAi plants with deformity stigma,indehiscent anthers were accumulated in the lower part of stigma;the MLinc20 RNAi plants showed the stigma exposed,anther dehiscent normally but pollen grains were very few;the MLinc22 RNAi plants have shorts filaments and indehiscent anthers.3.The single copy number of MLinc15 transgenic plants were multiplicationed,then the T3 homozygous lines were obtained.The expression of MLinc15 was significantly decreased in MLinc15-T3-4 and MLinc15-T3-19 lines.However,the expression of MLinc15 slight decreased in MLinc15-T3-3 and MLinc15-T3-23 lines.Then the MLinc15-T3-4 line and the MLinc15-T3-3 line and the control ‘Jin668’ were selected for phenotypic analysis.The results showed that the buds of MLinc15-T3-4 line were not full and the anthers were shriveled,and when the buds developed to blooming state,the length of buds(21 mm)was shoeter than that of ‘Jin668’(longer than 24mm).Subsequently,the cytological observation of anthers were performed,the result showed that the microspore development of MLinc15-T3-4 was advanced,and some microspores appeared deformity and obvious shrinkage in the early stage,and the tapetum degradation was faster than the control.The microspore shrinkage was more serious in the late stage anther,showing a strip shape.However,MLinc15-T3-3 and‘ Jin668’ did not appear obvious abortion phenotypes,indicating that reducing the expression of MLinc15 lead to cotton anther abortion,and the degree of abortion has a dose effect.4.Compared with the control,MLinc15-T3-4 showed obvious abortion phenotype at the early stage anthers.In order to explore the internal mechanism,transcriptome analysis was performed on the early anthers of MLinc15-T3-4 and ‘Jin668’.Subsequently,GO analysis was performed on the screened differentially expressed genes,the results revealed that the differentially genes were enriched in the pathways:oxidative stress response,jasmonic acid biosynthesis process,peroxidase activity etc.The results of q RT-PCR verification showed that the expression of all differential genes involved in jasmonic acid biosynthesis was significantly up-regulated in MLinc15-T3-4.5.In order to determine that effect of MLinc15-T3-4 activates the jasmonic acid biosynthesis pathway in anthers,we measured the anthers jasmonic acid content in the MLinc15-T3-4 and ‘Jin668’.The results showed that the content of jasmonic acid in the anthers of MLinc15-T3-4 was significantly higher than that of the control ‘Jin668’at the same developmental stage,indicating that MLinc15 may be involved in the regulation of cotton anther development by affecting jasmonic acid homeostasis.