| Plant growth and development is an extremely complex process,starting from seed germination,undergoing seed growth and development,flowering,pollination,fertilization,fruiting,aging and death.Plants will suffer from a series of external stresses during growth and development.In order to adapt to the adverse environmental conditions,plants have evolved complex regulatory networks to reduce the damage caused by external stresses.As an important plant hormone,cytokinin plays an important role in this regulatory network.Cytokinin is a kind of adenine derivative,which participates in cell division and differentiation,bud formation and bud growth,as well as seed germination and flower and fruit development.Cytokinin oxidases/dehydrogenases?CKXs?catalyze the irreversible degradation of CTKs and participate in a series of growth and development processes of plants.It is the only known enzyme that specifically degrades CTKs.Many studies have shown that the expression levels of CKXs are induced by drought stress,and overexpression of CKXs increase plant tolerance to abiotic stress and affect plant development.In addition to being able to participate in plant abiotic stress responses,the CKXs genes also affect plant development.In apple,there are still few studies about CKXs.In this study,we first identified 10 MdCKXs genes from the apple genome database,and carried out phylogenetic tree analysis and bioinformatics analysis.We then analyzed the abiotic stress responses of these apple CKX family members by qRT-PCR,and found that MdCKX7.2?the gene sequence number:MDP0000279125?showed significantly higher expression than other members in response to ABA.Therefore,MdCKX7.2 was selected as the main research object.MdCKX7.2 was cloned from the genome of Malus domestica Borkh,and its promoter sequence was analyzed.The promoter analysis of MdCKX7.2 gene revealed that there were multiple stress responsive elements in the MdCKX7.2 promoter sequence,suggesting that it may be involved in the response of plants to adverse environments.In order to study the biological functions of MdCKX7.2,we obtained MdCKX7.2 transgenic Arabidopsis thaliana and tested the response of drought,ABA and low phosphorus in transgenic Arabidopsis thaliana.The test results are as follows:1.Bioinformatics analysis of MdCKX7.2 geneMdCKX7.2 was 1542 bp in size and encoded 513 amino acids.Gene structure analysis showed that MdCKX7.2 contained 5 exons and 4 introns,which were located on chromosome8 of apple.Protein conserved domain analysis indicated that MdCKX7.2 had a conserved FAD binding domain at the N-terminus and a conserved cytokinin binding domain at the C-terminus.2.Ectopic expression of MdCKX7.2 gene in Arabidopsis enhanced its drought stress toleranceThe promoter sequence of MdCKX7.2 contained an MBS element associated with the drought response.Expression analysis revealed that the expression of MdCKX7.2 was significantly induced by PEG.Drought assays showed that the survival rate of MdCKX7.2transgenic plants was significantly higher than that of wild-type controls.The expression levels of stress response related genes such as RD22 and RD29B in transgenic Arabidopsis were significantly higher than those in wild type.These results indicate that the ectopic expression of MdCKX7.2 enhances the resistance to drought.3.MdCKX7.2 is involved in ABA-mediated seed germinationThe promoter analysis revealed that there was an ABRE element associated with the ABA response.Real-time quantitative PCR analysis revealed that the expression of MdCKX7.2 was significantly induced by ABA.After treated with 1?mol·L-11 ABA,the seed germination rate and fresh weight of MdCKX7.2 transgenic Arabidopsis were significantly decreased compared with wild type.Meanwhile,the expression levels of EM1,EM6,and RD29B were significantly up-regulated in transgenic lines.It is indicated that ectopic expression of MdCKX7.2 in Arabidopsis increases the sensitivity to ABA in plants.4.MdCKX7.2 is involved in the absorption and utilization of phosphorus in plantsReal-time quantitative PCR analysis revealed that the expression of MdCKX7.2 was significantly induced by low phosphorus stress.Wild-type and MdCKX7.2 transgenic Arabidopsis were treated with low-phosphorus(40?mol·L-1)medium.The results showed that the primary root length of MdCKX7.2 transgenic Arabidopsis seedlings was significantly longer than that of wild-type controls.Similarly,the fresh weight increased and the proline content increased in transgenic lines.These results indicate that ectopic expression of the MdCKX7.2 in Arabidopsis increased its tolerance to low phosphorus. |
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