Cloning And Functional Analysis Of MxWRKY106 Gene From Malus Xiaojinensis

In this study,the Apple iron-efficient genotype Malus xiaojinensis Cheng et Jiang was used as the test material.The specific primers were designed by homologous cloning method,and the cloned WRKY transcription factor was used as the research object.The RNA of Malus xiaojinensis was extracted and reverse transcribed into single-stranded c DNA,and the MxWRKY106 gene was amplified by PCR.The open reading frame of MxWRKY106 gene is 1059 bp in length,encoding 351 amino acids,contains only one WRKY domain,and the zinc finger structure is C 2H2,indicating that it belongs to class II of the WRKY family.Using the software MEGA7 to construct a phylogenetic tree of the MxWRKY106 gene and homologous genes in other species,it was shown that MxWRKY106 has the highest homology with apple(Malus domestica)Md WRKY53.The results of subcellular localization showed that the MxWRKY106 protein was localized in the nucleus.The results of semi-quantitative RT-PCR showed that the expression of MxWRKY106 gene in different parts of Malus xiaojinensis was organ-specific.In the normal Hoagland nutrient solution culture(0h),the MxWRKY106 gene was expressed in the extracted organs and the expression amounts of new leaves and roots were highest.Under low temperature stress(2°C),salt stress(200 m M Na Cl),iron deficiency stress(4 ?M Fe-EDTA)and iron excess stress(160 ?M Fe-EDTA),as time went on,the expression levels of MxWRKY106 increased first,then decresed.Real-time quantitative PCR analysis showed that under normal Hoagland nutrient solution culture conditions(0h),the expression amount of MxWRKY106 gene in different parts was the highest in the new leaves,followed by the roots,and the old leaves were slightly higher than the phloem.Under low temperature(2°C),high salt(200 m M Na Cl),iron deficiency(4 ?M Fe Na-EDTA)and iron excess(160 ?M Fe Na-EDTA),In the new leaves of Malus xiaojinensis,the expression level of MxWRKY106 gene increased first and then decreased from 1h to 24 h after treatment.The peak was reached after 3h of low temperature treatment,6h of high salt treatment,9h of iron deficiency treatment and 9h of iron excess.The expression of MxWRKY106 gene also showed a trend of increasing first and then decreasing in roots of Malus xiaojinensis,and reached the peak at low temperature,high salt,iron deficiency and iron excess at 9h,3h,6h and 6h,respectively.The Agrobacterium tumefaciens-mediated MxWRKY106 gene was overexpressed in Arabidopsis thaliana,then obtained three transgenic lines.After 7d of salt treatment,wild-type Arabidopsis showed significant yellowing compared with transgenic;After 14 d of iron deficiency treatment,the wild-type Arabidopsis thaliana was dwarf and had fewer roots,while the transgenic plants grew normally and only a few leaves turned yellow;After 14 d of iron excess,the wild-type Arabidopsis leaves were curled and the leaves were purple,while the transgenic plants only old leaves yellow and the new leaves grew normally.The measured physiological indicators related to plant resistance showed that the activities of CAT,SOD,POD and proline content in transgenic Arabidopsis thaliana were higher than those in the untreated group,and the MDA content was not changed compared with the control group.Large,chlorophyll content decreased,and the difference was large compared with the control group.It indicated that the MxWRKY106 gene transgenic Arabidopsis plants were less damaged than wild type after treatment with high salt,iron deficiency and iron excessive,and the resistance to high salt,iron deficiency and iron excessive stress was enhanced.In conclusion,overexpression of the MxWRKY106 gene enhances the tolerance of Arabidopsis to high salt,iron deficiency and excess iron stress.