| The growth and development of plants are easily regulated by environmental factors,among which high temperature(heat stress)conditions are a very common abiotic stress factor that has a great impact on plants.High temperaturew will cause the plants to suffer from water shortage and wither or even die,and will also cause a series of adverse effects such as the reduction of the production of main crops.Therefore,with the support of modern biotechnology,genetic engineering methods are used to improve the plant's resistance to stress and reduce the damage caused by high temperature to the growth,development and yield of plants,which has important research value and practical significance.In this paper,three phosphorus transporter genes(PTs)in potato that may have a positive effect on heat tolerance were firstly analyzed and screened through bioinformatics methods,and their functions were identified in Arabidopsis,tobacco and yeast.Then,using the method of potato transformation mediated by Agrobacterium,the effects of the three genes on heat resistance of potato were analyzed.Finally,through the detection of some relevant physiological indicators,and the use of yeast double-hybrid technology and bimolecular fluorescent complementation(BIFC)technology to screen interacting proteins,the physiological and bioc hemical and molecular mechanisms of these three PTs to improve the heat resistance of potato were initially explored.The main results are as follows:(1)We identified 22 PTs from the protein database of potato.These 22 PTs can be divided into 6 subfamilies.We have analyzed and explained their gene/protein properties,gene duplication events,etc.Among them,the PHO1 subfamily has the longest protein sequence and the largest molecular weight.GO annotations show that this family is an essential membrane component and is the only hydrophilic protein family.At the same time,the transcript data obtained from the PGSC database and quantitative experiments show that St PHO1.1,St PHO1.2 and St PHO1.3(collectively named St PHO1s)have an effect on the heat resistance of potatoes.(2)Construct the overexpression vector p BI121 and yeast expression vector p YES2 of St PHO1 s,and then introduce them into Arabidopsis and inject tobacco,and transform them into yeast,respectively.Through heat resistance experiments,we found that with the transfer of St PHO1 s and p BI121 or p YES2 recombinant plasmids,the heat resistance of Arabidopsis,tobacco and yeast have been improved to a certain extent,which showed that St PHO1 s does have an important role in improving heat resistance.(3)The overexpression recombinant plasmid of St PHO1 s was introduced into the wild-type tetraploid potato "Desiree",and the transgenic plant with St PHO1.1,St PHO1.2 or St PHO1.3 was successfully obtained: St PHO1.1-Desiree,St PHO1.2-Desiree and St PHO1.3-Desiree.Heat resistance experiments show that when nutrients are sufficient,the phenotype of transgenic plants(especially St PHO1.2-Desiree and St PHO1.3-Desiree)under high temperature stress is better than that of wild type,indicating that St PHO1 s is also important for improving the heat tolerance of potatoes.(4)The detection of related indicators showed that the POD,CAT and APX activities of transgenic potatoes were significantly higher than those of the wild type,and the soluble sugar content was also significantly higher than that of the wild type.The chlorophyll-a and-b and carotenoid content in St PHO1.1-Desiree and St PHO1.3-Desiree were higher than wild-type plants,but the content of three chloroplast pigments in St PHO1.2-Desiree was almost unchanged compared with wild-type plants.Moreover,the photosynthetic rate,proline content and relative conductivity(REC)of the three-gene transgenic plants had no obvious advantages.(5)We analyzed the subcellular localization of St PHO1 s and found that they are mainly expressed on the cell membrane,while a small amount of expression is also present in the nucleus or cytoplasm.Using yeast double-hybrid technology,a number of interacting genes/proteins were screened from Arabidopsis library plasmids,and corresponding genes/proteins were matched in potato.Through the “one-to-one” identification of cotransformed yeast,we obtained several important interaction genes/proteins,including glutaredoxin(Glu,interacts with St PHO1.2)and rubredoxin(Rub,interacts with St PHO1.3),the protein PSII related to photosynthesis(interacts with St PHO1s),the protein Ca2 related to calcium ion transport(interacts with St PHO1s),and the member of the heat shock protein 40 family DNAJ(Hsp,interacts with St PHO1.1).We choose Ca2 and PSII,which interact with St PHO1 s,and Hsp,which interacts with St PHO1.1,for subcellular localization and BIFC verification.The results showed that Hsp,Ca2 and PSII were located in the cell membrane and cytoplasm,and the interaction of St PHO1.1 and Hsp(DNAJ),the interaction of Ca2 and PSII with the three genes all occurred in the cell membrane and cytoplasm.In conclusion,our results suggested potato genome contained 22 PTs,which were divided into 6 subfamilies.Among them,St HO1.1,St PHO1.2 and St PHO1.3 of the PHO1 subfamily played an important role in improving the heat tolerance of plants.Detection of physiological indicators and analysis of interacting proteins indicated that these three genes may improve plant heat tolerance through increased antioxidant enzyme activity,soluble sugar content,and interaction with photosynthetic and calcium ion transport proteins.In addition,heat shock proteins are a family of proteins that are very important for plants to resist high temperature stress.The interaction between St PHO1.1 and DNAJ(a member of the Hsp40 family)also showed that St PHO1.1 might also interact with DNAJ to regulate a series of other physiological and biochemical reactions,thereby improving the heat resistance of plants. |
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