| Boron pollution in boron-rich soil and boron-related enterprises in arid and semi-arid regions have serious negative effects on the growth and development of plants and animals.Previous studies by foreign scholars and our research group have shown that poplar can enrich boron in leaves,which is suitable for bioremediation of boron-rich soil.However,very little research has been done on the molecular aspects of the woody poplar.Our research group has analyzed the expression patterns of boron transporter family(BOR)in different tissues under boron stress,and screened and cloned the key genes of BOR.In this experiment,boron-tolerant characteristics of Populus russkii and Populus tomentosa were analyzed.The boron transport and distribution and enrichment processes of BOR were studied by constructing over-expression vector and RNA interference vector to convert Populus tomentosa.The tissue specificity of BOR gene promoter and its response mode to boron stress were studied by constructing BORpro::GUS transformation into Populus tomentosa.Subcellular localization in poplar BOR were analyzed by transforming arabidopsis thaliana.The research results obtained are as follows:1.Boron tolerance of Populus russkii and Populus tomentosa and the expression pattern of BOR family.Different boron concentration gradients(CK-50 um,1m M,5m M,10 m M)were applied to the hydroponic seedlings of Populus russkii and Populus tomentosa.The results showed that the leaves of Russian poplar turned yellow and curled after a week of 10 m M boron stress,while the leaves of Populus tomentosa showed a large number of black spots after 4 days under 10 m M boron stress.Through the determination of boron content,boron was mainly concentrated in the leaves of plants for detoxification under high boron stress.In BOR family,Pr BOR7 and Pto BOR7 expression levels significantly increased with stress time under high boron stress.2.Function of Populus russkii BOR7 geneThe 35Spro:: Pr BOR7 vector constructed in this study was converted into Populus tomentosa by the leaf disconversion method.Thirty poplar trees were cultured in tissue culture,among which 23 were positive,with a positive rate of 76.67%.Through real-time fluorescence quantitative PCR analysis,three lines with high BOR7 expression were screened out NO.1and NO.13 and NO.19,with the expression levels 80,40 and 50 times higher than those of wild-type poplar,respectively.High boron treatment was carried out on poplar with heterogenous Pr BOR7 gene expression.In the experiment of hydroponic culture,the treatment of 4 different boron concentration gradients(CK-50 um,1m M,5m M,10 m M)was designed.The results showed that on the third day of boron stress of 10 m M,the transgenic poplar was the first to have a small amount of necrotic black spots than the wild-type poplar.On the fourth day,the transgenic poplar had more black spots than the wild-type under 10 m M of boron stress,and the trans genic poplar had more black spots than the wild-type under 5m M of boron stress.In the determination of boron content,there was more bo ron in the leaves of overexpressed lines than that of wild-type leaves.The results showed that the transgenic plants absorbed boron faster,which led to the occurrence of boron stress phenotype earlier than the wild type.Low boron treatment was performed on poplar with heterogenous Pr BOR7 gene expression.After treatment with low boron concentration(5u M)for one month,it was found that transgenic poplar grew higher a nd budded faster than wild-type plants.Over-expressed lines was significantly higher th an that in the wild type in the bark and bud parts,indicating that boron content affected the growth of plants,and it was speculated that Pr BOR7 might have boron absorption function.3.Construction of interference vector and screening of transgenic seed lings Firstly,interference expression vector was constructed,and more than 100 tissue culture poplar trees were obtained after the transformation of Populus tomentosa.Among them,80 were positive poplar trees,with a positive rate of about 80%.Through real-time fluorescence quantitative PCR analysis,the three lines with the lowest BOR7 expression--NO.41 and NO.70 and NO.76--were screened out.4.GUS staining and subcellular localization of BOR7 The plant expression vectors Pr BOR7pro::GUS were transformed into populus pubescens.The tissue expression differences of Pr BOR7 promoter transgenic were analyzed.Preliminary results showed that Pr BOR7 was mainly expressed in stem and old leaf,but not in root at normal level.In the protoplasts of arabidopsis thaliana,the transient green fluorescent protein showed that the main sites of Pe BOR7 were on the cell membrane and nucleus... |
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