Cloning And Regulation Of PIN1 Gene Among Different Development Stages Of Swollen Stem In Zizania Latifolia

Due to the infection and reproduction of Ustilago esculenta,the development of the stem were changed to form the swelloen stem in Zizania latifolia,following with the inhibition of apical growth at stem.As the efflux carrier of IAA polarity transport,the PIN1 played an important regulatory role in plant growth and development,including the development of embryo and vascular tissue,cell size,tropism,and formation of flower organ.In this study,the genes of Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c were cloned and their expression patterns among different stages of stem were elucidated in "Zhejiao No.7",which were related to the polarity transport of IAA.The induced expression were proceed after treatment with IAA and TIBA in in Z.latifolia.Based on the dynamics analysis of mycelium growth and distribution of U.esculenta in stems,the regulation of Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c among different development stages and phenotypes of swollen stem were discussed in Z.latifolia.After comparative analysis of U.esculenta in vitro and cultivation of Z.latifolia in field,the effects of IAA and TIBA on the mycelium growth of U.esculenta and the production of cultivation were investigated.The regulation mechanism of TIBA treatment in the promotion of cultivation were discussed,which was related to the regulation of IAA polarity transport.Besides,the over-expression of Zl-PIN1 were proceed in Z.latifolia by the methods of genetic transformation,which would provide technical support for function research of genes in Z.latifolia.The results were as follows:1.Full length cloning of Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c genes in Z.latifoliaThe full-length sequences of Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c genes were cloned,which had the special domain of Mem-trans in the auxin efflux carrier protein of PIN1.The full-length DNA of Zl-PIN1 was 2 408 bp and its c DNA is 1 770 bp,encoding 589 amino acids,containing 6 exons and 5 introns.It was closely related to indica and its amino acid sequence similarity is 96%.The full length of Zl-PIN1 b DNA was 2 024 bp and its c DNA is 1 563 bp,encoding 526 amino acids,containing 4 exons and 3 introns,It was closely related to wild rice,with amino acid sequence similarity of 87%.The full-length DNA of Zl-PIN1 c was 2 421 bp and its c DNA is 1 797 bp,encoding 598 amino acids,containing 6 exons and 5 introns,It was closely related to wild rice with amino acid sequence similarity of 95%.Phylogenetic analysis showed that the genes of Zl-PIN1 and Zl-PIN1 c could be clustered into one group,with far distance with Zl-PIN1 b,which might be related to the different structure of predicted trans-membrane helix region.2.Expression patterns of Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c genes in Z.latifoliaThe expression patterns of Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c genes were proceed among three phenotypes of stem in Z.latifolia,which might be related to the morphological changes of stem.The expression levels of three genes in white Z.latifolia and gray Z.latifolia stems were significantly lower than that in male Z.latifolia at the early stage of swollen stem(P<0.05).There was no significant difference in the expression of three genes before jointing(stages of 5-leaf and 8-leaf),but the expression was significantly increased after joint development of stem in male Z.latifolia(P<0.05).It was suggested that Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c may be involved in the regulation of the apical growth related to jointing of the stem.The similar expression patterns were found during development of swollen stem between Zl-PIN1 and Zl-PIN1 c,which were significantly different from Zl-PIN1 b.The expression of Zl-PIN1 and Zl-PIN1 c gradually increased during development of white Z.latifolia and peaked at 10 cm after swelling,and then decreased significantly.whereas,the expression of Zl-PIN1 b gene gradually decreased during the early stage of development,and slight increased at 10 cm after swelling,then significantly decreased(P <0.05).There were differences in the expression patterns of the three genes between grey Z.latifolia and white Z.latifolia.The expression of Zl-PIN1 and Zl-PIN1 c increased gradually during the development of swelling in grey Z.latifolia,with the significantly higher expression than that in white Z.latifolia at the late stage of swelling.However,the expression levels of Zl-PIN1 b gene gradually decreased,and did not express at 10 cm after swelling,which could be related to the significant differences in the mycelia infection and growth distribution between the two phenotypes of swollen stem.The expression of Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c in stem of Z.latifolia could be regulated by treatment of IAA and TIBA,with the significant dose effects.The induced expression of Zl-PIN1 and Zl-PIN1 c could be increased by IAA treatment in male Z.latifolia,while no significant induction were found in Zl-PIN1 b.The expression of the three genes were decreased after treatment with IAA in white Z.latifolia,which was similar to the treatment of higher concentration of TIBA.Based on the inhibition of mycelium growth in stem after treatment with TIBA,the induced expression of Zl-PIN1,Zl-PIN1 b and Zl-PIN1 c in white Z.latifolia may presumably be related to the mycelium growth of U.esculenta in stem.3.Effects of hormone on the culture and mycelium growth of U.esculentaThe mycelium growth of U.esculenta in vitro showed that IAA could promote the mycelium growth in the T and MT strains,with positive correlation with the concentration of IAA.Low concentrations of TIBA treatment could promote the mycelium growth of U.esculenta,but the high concentrations could inhibit the mycelium growth both in the T and MT stains,with significantly reduced in the dry weight of haploid strain.There were difference in the tolerance to TIBA treatment between T-and MT-type strains.Treating with TIBA at 10 ?M,the mycelium growth were promoted in MT-type stains,but inhibited in T-type stains.While treating with TIBA at 50 ?M,the dry weight of MT-type strains reduced significantly while no effects were found in T-type strains.Besides,the treatment with 6-BA could promote the mycelium growth of T-type with the similar to IAA treatment.Low concentrations of 6-BA(1-5 ?M)could promote the mycelium growth of MT-type,but high concentrations of 6-BA could inhibit the mycelium growth,which was similar to the treatment of TIBA.4.Effects of hormone on the cultivation of Z.latifolia in fieldThe harvest and yield of Z.latifolia could be effectively regulated by the treatment of hormone in the field.The harvest time could be delayed about nine to twelve days by treating with IAA,with no effects on the yield in Z.latifolia.However,the harvest time could be promoted by treating with TIBA,with increasing yield in the early stage of harvest.The yield of Z.latifolia could be increased in the middle stage of harvest by treating with 6-BA.Based on the analysis of economic characters in Z.latifolia,it was found that harvest tillers with swollen stem could be significantly increased by treating with TIBA and 6-BA.All of the three treatments had no significant effects on the other characters,liking weight of shell and net,length and coarseness of swollen stem,height of plant and leaf.Combined with the effects on the growth of U.esculenta,the regulation of TIBA might be related to the effects of U.esculenta and polarity transport of IAA in Z.latifolia,which could promote the effective distribution of nutrient between development of swollen stem and plant growth in Z.latifolia.5.Establish of overexpression genetic transformation system with Zl-PIN1 in Z.latifoliaThe overexpression genetic transformation vector of p FGC-Egfp-Zl-PIN1 was constructed.Based on the infection of agrobacterium tumefaciens,the method of transformation at growth point in stem was used to establish the transformation in wild Z.latifolia.Ten transgenic plants were identified by PCR detection.By the observations of GFP fluorescence signals,the localization of Zl-PIN1 protein was found at the bottom of the membrane among the perivascular cells,which was consistent with the result of Os PIN1 localization.The Zl-PIN1 overexpression transgenic plants were initially identified and the conversion rate was 3.7%.