Identification And Functional Analysis Of The MADS-box Gene In Endangered Mangrove Lumnitzera Littorea

Lumnitzera littorea(Jack)Voigt is a species of mangrove plant in family Combretaceae.It is listed as endangered species and in China it is found only in Hainan Island.Based on the previous investigation,we found that there were two flowering patterns in L.littorea,one was stigma stretching out of the petals before petals unfolding,and another one was stigma and stamen stretching concurrently after petals unfolding.The occurrence of the synchronous difference in development of stamen and pistil is closely related to the differential expression of genes regulating the floral development.Thus the transcriptome sequencing of the floral organs of these two flowering patterns was performed.The MADS-box gene family is closely related to floral organ development.These genes not only play an important role in regulating the process of flowering time process and flower organ development,but also participates in the regulation of plant thermomorphogenesis.In this study,firstly the results of high-throughput sequencing of two flowering patterns were analyzed,and 12 candidate genes of MADS-box were screened by bioinformatics analysis.Secondly,the candidate genes were cloned,their over-expression vectors were constructed and they were transformed into Arabidopsis thaliana.The phenotype of A.thaliana with transformed genes was observed and the functional characterization of MADS-box gene was analyzed.The main results are as follows:1.Based on the previous investigation of our research group,it was studied the transcriptome profiles at two different flowering patterns and the differentially expressed genes of L.littorea were screened by combing with the bioinformatics analysis,the sequences of MADS-box gene of Arabidopsis thaliana,Hevea brasiliensis and Fragaria ananassa were compared with those of L.littorea,and 50 MADS-box genes were identified.Twelve candidate genes were screened by qRT-PCR,and their ORF,homologous genes and functions were predicted.2.12 candidate genes were amplified to construct over-expression vector.Then 4 genes overexpression vectors were transformed into A.thaliana.Finally,the transgenic homozygous seeds were obtained by screening and continuous planting.3.The function of LlMADS1 was analyzed under optimal temperature(22?)and themild high temperature(28?)for A.thaliana.The gene sequence of LlMADS1 is highly similar to AP3.The abnormal development of the petals and stamens was observed after OX-LlMADS1 transgenic plants flowering.Therefore,the LlMADS1 gene is mainly involved in the regulation of petals and stamens development.4.Under the condition of optimal temperature(22?)and the mild high temperature(28?)for A.thaliana,the function of LlMADS11 was analyzed.Through the phenotypic observation of OX-LlMADS11 transgenic Arabidopsis thaliana,it was found that LlMADS11 could control plant flowering time in A.thaliana.Respond to high temperature and change the plant morphology in petiole elongating,leaf thinning and florescence advancing.Therefore,it is suggested that LlMADS11 gene is involved in plant thermomorphogenesis.As homologous sequence of LlMADS11 has not been found,its characteristics were unknown.Ll MADS11 was mainly located in the nucleus by using Cam35S-GFP as expression vectors in the subcellulars positioning of LlMADS11.5.The temporal and spatial expression of LlMADS1 and LlMADS11 in five tissues of L.littorea was analyzed.The results showed that the expression of LlMADS1 was relatively high in blooming flowers,and the highest expression LlMADS11 was in the flowers with the stigma stretching out of the petals before petals unfolding.