| About three-fourths of the cultivated land in our country are in lack of phosphorus,which has become one of the main factors restricting the yield of maize.It is one of the effective ways to improve maize yield by exploring low phosphorus resistant gene resources and using molecular marker assisted selection and transgenic technologies to cultivate maize with high efficient phosphorus.In our previous study,three candidate genes,ZmWRKY101,ZmMYB015 and ZmSPX-MFS1 were found respond significantly to low phosphorus stress in maize roots.Here,the genetic transformation of the three genes was carried out in rice background via by Agrobacterium tumefaciens mediated method,the phenotype and the function of each gene in response to low phosphorus stress were preliminarily verified.The main results are as follows:1?The genes ZmWRKY101,ZmMYB015 and ZmSPX-MFS1 in maize were cloned and sequenced,respectively.In-Fusion technique was used to connect each gene sequence to PHB vector,containing kanamycin and hygromycin resistant genes and two tandem copies of 35 S CMSV promoter,and the overexpression vector was successfully constructed.2?By using Agrobacterium mediated method and applying rice cultivar Nipponbare as transformational background materials,the overexpression vectors containing target genes were introduced into Nipponbare,respectively.The screening of hygromycin resistance,PCR positive detection and DNA sequencing results showed that the target genes had been integrated into the genomes of Nipponbare,and the transgenic rice plants of ZmWRKY101,ZmMYB015 and ZmSPX-MFS1 genes were achieved,respectively.The t1-generation positive conversion rate of the 3 genes in PCR was 14.28%,34.32% and 67.14%,respectively.3?Identification and root system screening of T2 transgenic generation rice seedlings with over expressing target genes were carried out under low phosphorous in hydroponic cultures stress.The average root length(RL),root surface area(RSA),root volume(RV),root tip number(RTN),and branch number(BN)of plants with overexpressed one of the three genes under low phosphorus stress,showed significant difference between the negative and the wild type plants,and the over expressed transgenic plants have thicker root systems under low phosphorus stress.The results showed that over-expression of these 3 exogenous maize genes could significantly change the root architecture of rice and enhance the tolerance of low phosphorus stress in transgenic plants.4?The real time fluorescence quantitative(RT-PCR)analysis of ZmMYB015,ZmSPX-MFS1 and ZmWRKY101 gene overexpressed plants under low phosphorous stress showed that the relative expression level of the positive transgenic rice plants was much higher than that in the negative control(CK,Nipponbare of wild type),respectively.The expression level of ZmWRKY101,ZmMYB015 and ZmSPX-MFS1 transgenic plants was about 25 to 40 times,40 to 90 times and 8 to 25 times higher than that of CK,respectively.The results further showed that all the three genes were highly expressed in rice under low phosphorous stress.5?Correlation analysis was performed between phenotypic data and relative expression level of target gene transgenic plants,respectively We found that the expression level of ZmMYB015 was significantly correlated with RL(P=4.632E-6),RSA(P=1.330E-2),RTN(P=8.632E-3)and BN(P=8.150E-4)of ZmMYB015 transgenic plants,indicating that the plants with higher expression level of ZmMYB015 has longer RL,larger RSA,and more RTN and BN.The RL(P=1.298E-4),RSA(P=6.525E-3),RV(P=1.631E-2),and BN(P=1.080E-3)of ZmWRKY101 gene positive transgenic plants have significant correlation with the expression level of ZmWRKY101.It shows that the plant with higher expression level of ZmWRKY101 has more developed root characteristics.However,there was no significant correlation between root traits and the expression data of ZmSPX-MFS1 in ZmSPX-MFS1 positive transgenic plants. |
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