| HD-Zip transcription factors are widely distributed in the plant kingdom,from algae to angiosperms.HD-Zip transcription factors are involved in plant’s growth and development,these transcription factors also help plants fight against abiotic stresses.Brassica napus L.(2n=38,AACC genome)possibly arose from interspecific hybridization between Brassica rapa(2n=20,AA genome)and Brassica oleracea(2n=18,CC genome),both B.rapa and B.oleracea are diploid,while B.napus is allotetraploid.B.napus is an important kind of oil crop,the researchers also regard it as a model plant when they study about heteroploid polyploidy.In this study,HD-Zip genes were identified and systematically analyzed in B.napus.We also try to explore the function of HB7 and HB12 of B.napus.The main results are as follows:1.A total of 165 HD-Zip genes were identified in the whole genome of B.napus,and the obtained genes were divided into 4 subfamilies based on the phylogenetic tree branch and the classification criteria of Arabidopsis thaliana HD-Zip genes.HD-Zip Ⅰ-Ⅳ subfamilies contained 69,26,13 and 57 proteins,respectively.There are a total of129 sister gene pairs at the branch end of the evolutionary tree,including 63 An-Ar orthologous gene pairs and 39 Cn-Co orthologous gene pairs.It indicated that the HDZip genes in the An and Cn subgenomes of B.napus were more closely related to their corresponding ancestral species.2.By analyzed the conserved motifs of HD-Zip proteins,we found that the conserved motifs in subfamily I and II were similar,and the types and numbers of motifs are much smaller than those of subfamily III and subfamily IV.Motif2 and motif1 basically correspond to the Homeodomain,and motif6 basically corresponds to LZ(leucine zipper).Through the analysis of gene structure,it was found that the number and length of introns of subfamily III and IV were superior to those of subfamily I and II.Homologous genes belonging to the same branch in the evolutionary tree were quite similar both in gene structures and conserved motifs.3.By analyzed the HD-Zip collinear genes of A.thaliana,B.rapa,B.oleracea and B.napus,it was found that more HD-Zip domain lost events happened during the whole genome triplication of B.oleracea.And B.napus also lost some of its HD-Zip genes.4.We analyzed the cis-acting elements of Bn HD-Zip genes.It can be found that the light-responding elements ranked first for the quantity of its types.It’s distributed on the upstream of all HD-Zip genes.The tissue-specific elements ranked lowest,only8 tissue-specific elements existed on the upstream of Bn HD-Zip genes.There is no tissue-specific element found on the upstream of subfamily Ⅲ.Of all types of hormoneresponsive elements,the Me JA-responsive elements ranked first for the quantity of its types,but the ABA-responsive elements ranked first for its distribution ranges,we can find ABA-responsive elements on the upstream of 77.6% Bn HD-Zip genes.5.Transcriptomic data were used to analyze the expression patterns of Bn HD-Zip genes after B.napus treated with ABA,PEG and Na Cl,and it was found that Bna A.HB1.a,Bna C.HB1.a,Bna A.HB12.a,Bna A.HB12.b,Bna C.HB12.a,Bna C.HB12.b,Bna A.HB7,Bna C.HB7.a and Bna C.HB7.b were upregulated after treated with ABA;Bna A.HB12.a,Bna A.HB12.b and Bna C.HB12.a were upregulated after treated with Na Cl;Bna C.HB12.a was upregulated after treated with PEG.We collected the transcriptomic data of seeds at the 2,4,6 and 8 weeks after pollination(WAP)from B.napus,and found that HD-Zip genes were differentially expressed in seeds at different developmental stages.We divided the post-pollination time into three periods,“2-4WAP”,“4-6WAP” and “6-8WAP”,and 49(29.7%)Bn HD-Zip genes show their expression pattern as “up-down-down” and this kind of expression pattern ranked first.We analyzed transcriptomic data of seed germination process,and found that 15 genes were up-regulated at 12 h after imbibition(hai),43 genes were up-regulated at36 hai,63 were up-regulated at 72 hai,and only 4 genes were down-regulated,namely Bna C.HB7.b,Bna A.HAT3,Bna C.HDG12.a and Bna A.HDG1.b.6.Three homologs of the HB7 gene and three homologs of the HB12 gene of B.napus variety "Zhongshuang 11" were successfully cloned and named as Bn HB7.1ZS,Bn HB7.2ZS,Bn HB7.3ZS,Bn HB12.1ZS,Bn HB12.2ZS and Bn HB12.3ZS,respectively.The results of RT-q PCR experiments showed that those 6 genes’ expression level differ greatly in specific plant tissues,and up-regulated their expression level significantly after treated with ABA,PEG and Na Cl.7.Subcellular localization experiments showed that three homologs of HB7 and three homologs of HB12 all located in nucleus,which conformed to the localization characteristics of general transcription factors.Yeast-two-hybridization experiments showed that Bn HB12.2ZS and Bn HB12.3ZS could form self-homodimers,while Bn HB7.2ZS and Bn HB7.3ZS couldn’t form self-homodimers;any two of the three HB12 homologs could form homodimers,while HB7 homologs cannot.Notably,of all24 heterologous combinations of HB7 and HB12,four combinations could form heterodimers,suggesting a cooperative relationship between HB7 and HB12 proteins.In this study,a total of 165 HD-Zip genes were identified in B.napus,which is the species with the largest number of identified genes in this family.The HD-Zip gene family is divided into four subfamilies,and members of the same subfamily often have similar exon-intron structures,motifs and protein structures.We analyzed transcriptomic data and find that only a few members of the HD-Zip gene family respond to ABA,Na Cl or PEG treatments,and all of them were from subfamily I.The experiment further proved that both HB7 and HB12 of B.napus variety(Zhongshuang11)were induced by ABA,Na Cl and PEG stress.More than 50% genes of II-IV subfamilies have highly expression level during the seed development process.38.2%of HD-Zip genes upregulate their expression level at 72 h of seed germination.Compared with HB12,HB7 couldn’t form homodimers,but they can form heterodimers with HB12.In this study,we lay a foundation for the further study of the function of the HD-Zip genes in B.napus,and also provides a theoretical basis for studying the mechanism of the HB7 and HB12 transcription factors in B.napus under drought and salinity stresses. |
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