Isolation And Identification Of Anther-enriched Genes In Chinese Cabbage

Anther development in Chinese cabbage is precisely regulated by a complex gene network,but its mechanism is still unclear.Anther development is regulated by a complex gene network,and the study of its molecular mechanism has an important impact on plant breeding.In this study,the DH line ’FT’ of Chinese cabbage was used as the test material,and the full transcriptome analysis technology was used to sequence the anther and mix(vegetative mass of four true leaves),respectively.A series of mRNAs,microRNAs(miRNA),long non coding RNA(lncRNA)and circular RNAs(circRNA)related to anther development of Chinese cabbage were identified,so as to construct competitive endogenous RNA(ceRNA)regulatory network.The main results are as follows:1.Through whole transcriptome sequencing of Chinese cabbage anthers and vegetative bodies,a total of 9055 differentially expressed mRNAs(5177 mRNAs are predominantly expressed in anthers),585 differentially expressed miRNAs(330miRNAs are predominantly expressed in anthers),1344 differentially expressed miRNAs(1230 lncRNAs are predominantly expressed in anthers),and lncRNA and165 differentially expressed circRNAs(62 circRNAs are predominantly expressed in anthers)were identified.2.This study established a ceRNA-miRNA-target gene regulatory network related to the development of Chinese cabbage anthers,which included 196 differentially expressed lncRNAs,17 differentially expressed circRNAs,55 differentially expressed miRNAs,and 49 differentially expressed miRNAs.Many important transcription factors,enzymes and regulatory proteins related to the development of anthers have been found in the network.These include AP2-like ethylene-responsive transcription factor(BraA01g001240.3C regulated by 27 DElncRNA,four DEcircRNA,and two DEmiRNA;BraA07g016770.3C regulated by nine DElncRNA,and ten DEmiRNA),ethylene-responsive transcription factor(BraA09g056770.3C regulated by three DElncRNA and one DEmiRNA;BraA08g016860.3C regulated by two DElncRNA and one DEmiRNA;BraA02g036660.3C regulated by four DElncRNA,two DEcircRNA,and one DEmiRNA),and phosphoenolpyruvate carboxylase(BraA04g029950.3C)regulated by ten DElncRNA and two DEmiRNA.Alongside,squamosa promoter-binding-like protein(BraA04g003010.3C and BraA06g043820.3C)regulated by 15 DElncRNA,one DEcircRNA,and 13 DEmiRNA,and nitrate reductase(BraA02g024390.3C)regulated by five DElncRNA,one DEcircRNA,and one DEmiRNA.3.Randomly selected 13 mRNAs,7 miRNAs(4 known miRNAs and 3 new miRNAs),7 lncRNAs and 7 circRNAs from the whole transcriptome data.The expression patterns on the anthers and vegetative bodies of cabbage show that the q RTPCR results are basically the same as the whole transcriptome sequencing results,which further confirms the reliability of the RNA-Seq data;The expression patterns of BraA05g041890.3C and BraA04g030880.3C in different tissues of floral organs were further analyzed,and it was found that the above two genes were specifically expressed in anthers.4.The promoters of BraA05g041890.3C and BraA04g030880.3C were cloned,and the promoter activity analysis vector was constructed respectively,and transformed into Arabidopsis plants by the dipping method,and 28 positive seedlings were obtained through hygromycin screening and PCR identification.And 36 strains.GUS staining was performed on the tissues of transgenic Arabidopsis,and it was found that BraA05g041890.3C and BraA04g030880.3C were anther-specific expression genes.