| Petunia hybrida is in the family Solanaceae and genus petunia.It’s widely used as important bedding or plotting flower in landscape architecture with various colours and long flowering season.Due to the short life cycle,easy cultivation,successful genome sequencing,clear genetic background and easy transform by Agrobacterium tumefaciens,it has been developed as a model plant in research on transgenosis and ornamental plants.It requires manual pinch repeatedly for many plants to increase branches with lush flowers to achieve the desired ornamental value which can greatly increase the production cost and thus restricts the development of industry.However,new multiple-branch Petunia hybrida cultivar is not only the key to efficient plant production but the significant material for plant branching development research,proiding reference to multiple-branch research on other plants.The research on Arabidopsis thaliana showes that WRKY71 can increase the branching of Arabidopsis thaliana by upregulating the branching development gene and plays a role in branching development by enhancing the transportation ability of auxin.Over-expression of AtWRKY71 results in an increase of branches of Arabidopsis thaliana,and the phylogenetic tree shows that WRKY28 is closely related to WRKY71.Therefore,we speculated that WRKY28 could play an important role in the regulation of plant branching.In this experiment,PhWRKY28 and PhWRKY71 were cloned and bioinformatially analyzed,transcription level of Petunia hybrida after NaCl and PEG drought treatment and its different tissues were ananlyzed,and the promoter sequences of Ph WRKY28 and Ph WRKY71 were also cloned and bioinformatially analyzed.The main findings are as follows:(1)On the base of the published petunia genome datebase,the specific primers were designed.The Petunia hybrida cDNA provides template to amplification the Ph WRKY28 and PhWRKY71 sequences,and bioinformatics analysis was performed.The PhWRKY28 CDS region is 924bp and encodes 307 amino acids.The PhWRKY28 82%、65.4%similarity with Nicotiana tomentosiformis,Capsicum annuum respectively.The molecular formula of this protein is C2875H4828N92401186S189,which contains Ala(A)is the most,316 numbers,accounting for 34.2%of total protein;the PhWRKY71 CDS region is 1005bp,encoding 334 amino acids,and the similarity with Nicotiana tomentosiformis、Capsicum annuum is 69%、67%.The molecular formula is C1633H2503N465O524S14,which contains a maximum of 47 serine Ser.,accounting for 14.1%of total protein.(2)The expression quantity of Ph WRKY28 and Ph WRKY71 in different tissues of roots,stems,leaves,axillary and apex of Petunia hybrida are analyzed by means of Real-time fluorescence quantitative reaction.We found that PhWRKY28 gene specific express in the stem.The lowest in the apex,and the expression level in the stem is about 1.72 times much than apex;while the expression of PhWRKY71 in axillary is significantly higher than in other tissues,and the expression level in apex is the lowest.The amount is as much as 2.5 times the apex.(3)The expression characteristics of two genes under drought stress of NaCl and PEG showed that the expression of PhWRKY28 is up-regulated after NaCl treatment for 12 hours,and the expression of PhWRKY71 is up-regulated 1.6 times after NaCl treatment for 12 hours,and the expression decreased after 24 hours.The expression of PEG is up-regulated after 12 hours treatment,and the expression level after 24 hours is down-regulated after 12 hours.(4)Cloning promoter of PhWRKY28 and PhWRKY71 from Petunia hybrida.By using bioinformatics software,we found that the two promoter not only contains eukaryotic gene promoter TATA-box、CAAT-box,but also has some cis-regulatory elements that are induced by stress,such as ARE,MYB,and phytohormone-related elements.PhWRKY28 has a regulatory element(CAT-box)associated with the expression of plant tissue meristems. |
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