| Pinus massoniana is a kind of coniferous gymnosperms,abundant in tropical and subtropical areas in south China.Because of its fast growth rate and high yield,P.Massoniana has become one of the main forest-planting species in south of China.Because the soil in southern China is more acidic and phosphorus deficiency is common,P.massoniana has developed a series of strong resistance to the environment in the long natural evolution process.Proteins containing the SBP domain play an important role in plant response to P stress.With further research,it was found that miR156 targeted regulation of SPL gene in response to P stress.However,there are few reports on related studies in P.massoniana.Therefore,the study on the function of miR156-SPL regulatory module of Masson’s pine under low phosphorus stress provides a theoretical basis for selecting highquality germplasm resources of P.massoniana.In this study,a single family of P.massoniana was used as experimental material,and the basic information and expression patterns of SPL family members were identified through the low phosphorus stress transcriptome obtained by the previous research,and the expression localization of PmSPL family members was verified by subcellular localization.In addition,according to the overexpressed Pma-miR156a tobacco obtained by our research group in the previous stage,the relevant physiological indexes of the tobacco under phosphorus stress and the expression levels of miR156a and its target genes in different tissue parts were determined,and the targeting relationship was verified by RACE technology,providing information for miR156 regulation of SPL gene in response to low phosphorus stress.The main research results are as follows:1.Changes of morphological and physiological indexes of P.massoniana seedlings under low phosphorus stressTotal root length,lateral root number,root to shoot ratio,acid phosphatase activity and chlorophyll content of P.massoniana seedlings treated with low phosphorus were detected at different time periods.The results showed that under low phosphorus stress,total root length,lateral root number,root to shoot ratio and acid phosphatase activity increased,while chlorophyll content decreased.These results indicated that P.massoniana activated phosphorus in soil to adapt to low phosphorus environment through root morphological changes and acid phosphatase secreted by roots.In addition,the decrease of chlorophyll content indicated that low phosphorus stress could inhibit the photosynthetic efficiency of P.massoniana.2.Bioinformatics and expression analysis of SPL gene family members of P.massoniana under low phosphorus stressEleven members of P.massoniana SPL gene family were screened out from the total transcriptome data of P.massoniana under low phosphorus stress obtained by our previous research group.The amino acid numbers of all members ranged from 190 to 1147;The relative molecular weight was 21.6-127.1 k D;In theoretical isoelectric point analysis,1member was less than 6.5 acidic,6 members were more than 7.5 basic,and the rest were between 6.5 and 7.5.The hydrophobic index was less than 0,indicating that PmSPLs were hydrophilic proteins.Subcellular localization prediction analysis showed that all members were located in the nucleus.In motif analysis,all members contained motif 1,2,3;The results of amino acid comparison showed that all of its members contained SBP conserved domain,which consisted of two zinc finger structures and a nuclear localization signal,and the nuclear localization signal coincided with the second zinc finger structure.By analyzing the expression patterns of SPL family members under low phosphorus stress,PmSPL5/7 was only expressed in roots.PmSPL3/8 was only expressed in stems and leaves,showing certain tissue specificity,and all members were differentially expressed,suggesting that this family member may play a key role in the response of P.massoniana to low phosphorus stress.it was found that some members of PmSPL family had certain tissue specificity,which suggested that tehse PmSPL family genes played a key role in t response to low phosphorus stress.3.Function and target site validation of Pma-miR156a overexpressed tobacco under low phosphorus stressThe tobacco with overexpression of Pma-miR156a obtained by our research group was treated with low phosphorus stress.The results showed that the root structure of overexpressed plants was more developed than that of wild-type plants,and the length of taproot,number of lateral roots and total root surface area were significantly increased.Compared with wild-type tobacco,anthocyanin content,acid phosphatase and mitochondrial H~+-ATPase activity were significantly increased(p<0.05).Tissue expression pattern analysis showed that miR156a was significantly up-regulated in roots and down-regulated in stems and leaves under low phosphorus stress.Nt SPL6,which had the highest homology with PmSPL6,was significantly down-regulated in the root.These results indicated that miR156 regulated root systems in response to low phosphorus stress and it was speculated that PmSPL6 was regulated by miR156a.Further,the 3’UTR region of PmSPL6 was obtained by RACE,and the binding site of miR156a was found.Combined with the analysis of the expression levels of both,it was indicated that miR156a negatively regulated PmSPL6 and inhibited its expression,and its response site was mainly located in the root.4.Cloning,subcellular localization and genetic transformation analysis of PmSPL6 of P.massonianaCombined with transcriptome data,the longest open reading frame of PmSPL6 was cloned and validated,and constructed into the fusion vectors pCambia35s-EGFP and pCambia1305 containing GFP green fluorescent protein,respectively.Subcellular localization of PmSPL6 in tobacco showed that it was localized in the nucleus and cell membrane.Overexpressing PmSPL6 plants were obtained by agrobacterium-mediated transformation into Benni’s tobacco,and the root growth of overexpressing PmSPL6 tobacco was significantly inhibited after phosphorus stress. |
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