Functional Study Of BoLH27 And BoSF21 On Regulation Of Leaf Curvature In Cabbage

The Development of leaf curvature can change the flat malleable of blade, thereby affecting the plant respiration and photosynthesis., The natural development of leaves curvature is a very important agronomic traits.in cabbage (Brassica oleracea var Capitata L) Cabbage is widely grown in the world. In the long process of evolution, the young leaves, rosette leaves and heading leaves have significant differences in morphology. Young leaves and rosette leaves are flat expansion growing, and the heading leaves form a huge head by curling upward and inward. Current research on plant leaves curvature is mainly focused on the Arabidopsis thaliana and have achieved some leaf curvature mutants for Arabidopsis thaliana by natural mutation screening, which revealed that bHLH transcription factor, TCP family genes and auxin response factors play an important physiological function in regulating leaf morphogenesis and development. The cabbage have the typical characteristic of naturally leaf curvature, so it’s an ideal material for the study of leaf curvature. However, there are few studies had been reported on leaf curvature of cabbage. Therefore, understand the genetic development of cabbage leaf curvature and find important genes that involved in the complex regulation process will provide new point for the study of the molecular mechanisms of cabbage leaf morphogenesis and development, And then proceed the genetic improvement for the breed character based on the molecular genetic breeding methods..In order to explore the important genes controll leaf natural curvature of cabbage, and understand function of these genes, we choosed mid-maturation and good cabbage 519 for materials, and screened and cloned influencing factor BoLH27 and BoSF21 involved in signaling transduction of leaf curvature by transcriptome technology and RT-PCR technique. In order to study their biology function, we construct the overexpression vectors and suppression vectors, and transform Cabbage 519 through Agrobacterium mediated transformation to verify the specific features of BoLH27 and BoSF21. The main results as follows:(1) Transcriptome analysis and screening differentially expressed genes in stem tip and leaf at different developmental stages of CabbageBased on cabbage leaf morphological differences on natural growth, We extracted total RNA of stem tip and leaf at rosette stage and heading stage of cabbage. Then the samples were sent to Biomarker Technologies (Beijing) for transcriptome sequencing analysis. After the sequencing used HiSeqTM 2000, each sample was Unigenes data assembled by Trinity software, and a total of 54209 Unigenes annotation information were obtained. Moreover, IDEG6 software analysis revealed that 2923 genes displayed differentially expression in stem tip and leaf at rosette stages,2307 genes displayed differentially expression in stem tip and leaf at heading stages. There were 652 differentially expression genes in stem tip of rosette stages and heading stages,1786 differentially expression genes in leaf of rosette stages and heading stages, and 488 genes showed differentially expression in stem tip and leaf of rosette stages and heading stages. Combined NT, NR, GO, COG, SwissProt, TrEMBL, KEGG and relevant databases, by comparing the gene log2 (RPKM of heading stage leaves/RPKM of rosette stage leaves), and in accordance with the principle that the significant difference of leaf expression is from high to low at heading stage,15 genes belonged to bHLH transcription factor and 11 genes belonged to auxin response factor among these differentially expression genes.(2) The gene expression analysis of BoLH27 and BoSF21 wia qRT-PCRIn order to explore the relevance between BoSF21 and BoLH27 with leaf curvature, the expression levels in various tissues of cabbage were studied by real-time fluorescence quantitative PCR. Tips and Actin2 as reference genes. The results showed that BoLH27 genes expression displayed a gradual increasing trend in stem tip and leaf from rosette stage to heading stage, but no obvious expression difference was observed in stem tip. The expression level was higher in heading leaves than rosette leaves, there was 185.5 times more transcription at heading stage than rosette stage of BoLH27 which was consistent with transcriptome analysis results. BoSF21 have low-level expression in rosette leaves, and have high expression levels in heading leaves. The variation of BoSF21 expression levels was significant. There was 65.33 times more transcription at heading stage than rosette stage of BoSF21. The variation trend in expression levels by real-time fluorescence quantitative PCR was aslo accord with the results of transcriptome analysis, which indicated that BoLH27 and BoSF21 maybe associated with the regulation of cabbage leaf curvature..(3) Cloning and bioinformatics analysis of Cabbage BoLH27 and BoSF21 geneAccording to the gene sequences obtained from transcriptome sequencing, and combined with Brassica Database and Bolbase, we designed specific primers for gene cloning, and obtained the cDNA coding sequences for BoLH27 and BoSF21. The full-length CDS of BoLH27 was 795 bp, which encod 264 amino acids, prediction theory molecular weight was 30387.05 Da, and isoelectric point was 4.72. The secondary structure analysis showed that BoLH27 protein was mainly composed of 27.65% of the α-helix,12.88% of β-folding and 59.47% of random coil. PROSITE and InterProScan online prediction results show that BoLH27 have the typical bHLH domains(50～99 aa), and accord with conservative 13E-16R, and the Leu23 corresponding key amino acid sites. Phylogenetic tree analysis displayed that BoLH27 was closest to AtbHLH27, and the similarity was 81%, and was little related to AtTCP14.The full-length CDS of BoSF21 was 1035 bp, encoding 344 amino acids. Predicted theoretical molecular weight was 38082.48 Da, and isoelectric point was 5.694. The secondary structure prediction indicates that BoSF21 protein was mainly composed of 34.9% of the α-helix,12.8% of the β-folding and 52.3% of random coil. The online prediction results showed BoSF21 was a transmembrane protein, and the transmembrane region was 115-137 amino acid segments. Molecular phylogenetic analysis displayed that BoSF21 was closest to BrSF21, and the similarity was 96%, and was little related to Ricinus communis and Gossypium arboreum.(4) functional identification of BoLH27 and BoSF21 in transgeneAccording to the full-length sequence of BoLH27 and BoSF21, we designed the primers with restriction sites. Combined with the multiple cloning site of plant binary vector PCAMBIA1300, expression vectors were constructed containing BoLH27 and BoSF21 forward pC35S-35S:senseBoLH27, antisense pC35S-35S:antiBoLH27 and forward pC35S-35S:senseBoSF21 and antisense pC35S-35S:antiBoSF21. The expression vectors were transformed into cabbage seedling hypocotyl through Agrobacterium mediated transformation. The transgenic cabbage plants were obtained after pre-incubation, infection, dark culture, callus differentiation, proliferation of adventitious buds, rooting, hardening and transplanting process. Then extracting genome DNA of transgenic cabbage and PCR results showed that the appropriate stripes were detected. Besides, the real-time fluorescence quantitative PCR analysis displayed BoLH27 and BoSF21 had different expressed levels in transgenic plants, which indicated that BoLH27 and BoSF21 were successfully integrated into the genomic DNA. Compared with the wild-type plants, morphology observed that the antisense BoLH27 plants grew better and exhibited advance into the heading stage, and leaf size was much larger than the wild type. Their leaf base didn’t possessed the petiole and with more leaf lobes. However, there are no obvious phenotypic in sense BoLH27 plants compared with widetype plants. Because the phenological phase of sense and antisense BoLH27 plants different with widetype paints,so the phenotypic trait failed to be analyzed. Therefore, we have gained seeds of To generation, we will further analysis the function of the gene in T1 generation materials.