| Wound signaling pathways for plant regeneration are still an emerging field,with most studies focusing on the simple model plants Arabidopsis thaliana and Physcomitrella paten and their functions in other species.Current studies have shown that WIND1 can promote dedifferentiation of Arabidopsis thaliana and Fraxinus mandshurica cells and to a certain extent promote the formation of adventient buds or adventient roots,while STEMIN1 and ATR can promote cell reprogramming and change cell fate in the absence of wound induction in the Physcomitrella paten.In this paper,the sequences of PhWIND1 and PhSTEMIN1 genes were cloned and the expression patterns of PhWIND1,PhSTEMIN1 and their homologous genes were analyzed,and the genetic transformation of petunias,Arabidopsis thaliana and tobacco was carried out by constructing PhWIND1 and PhSTEMIN1 overexpression vectors,and preliminary gene function exploration of the obtained transgenic materials was carried out,aiming to promote the study of trauma factors WIND1 and STEMIN1 in petunia homologous genes.MIR319 participated in various periods of plant development and was closely related to the development of plant leaves and flower organs,while after overexpression of MIR319 in petunias,the leaf aspect ratio of transgenic plants decreased,the leaf surface was wrinkled,the leaf explant adventitious bud regeneration ability was enhanced,and a series of phenotypic changes of flower organs were accompanied by the formation of corrugated folds at the edge of the corolla,the deepening and overlapping edges of the petals and the decrease in the area of the corolla.In order to explore the effect of MIR319 on flower color and other ornamental properties,this paper selected another commonly used ornamental plant of Solanaceae,Nicotiana alata,as the material to overexpress MIR319,and observed the ornamental traits of transgenic plants.The main research results are as follows:(1)Two pretraumatic response factors,were selected based on the transcriptome sequencing results of the original petunia parent PA.The phylogenetic tree homology analysis showed that two pretraumatic response factors had the highest similarity with At WIND1(AT1G78080.1),and Pp STEMIN1(LC042086.1).The cloned gene sequences were named PhWIND1 and PhSTEMIN1,respectively.And further bioinformatics analysis.(2)PhWIND1 was expressed in the roots,stems,leaves and flowers of Petpetas,indicating that PhWIND1 could participate in the whole process of tissue and organ formation.PhSTEMIN1 was expressed in the roots,stems and leaves of Petunia,but hardly expressed in flowers,suggesting that PhSTEMIN1 was mainly involved in the growth and development of roots,stems and leaves,but not in the growth and development of flowers(3)PhWINDs and PhSTEMINS genes were further cloned according to genome and transcriptome sequencing results.The results showed that PhWIND1 and PhWIND2 were up-regulated and PhWIND3 down-regulated,indicating that PhWIND1 and PhWIND2 could be induced at the early stage of trauma,and PhWIND1 was more sensitive to wound signals.In PhSTEMINs,wound signals rapidly up-regulated PhSTEMIN2 and PhSTEMIN3 at the initial stage,while only the decreased expression of PhSTEMIN1 at the post-traumatic stage indicated differences in the expression of PhSTEMIN1 and its homologous genes.(4)The overexpression vectors p G-PhWIND1 and p G-PhSTEMIN1 were constructed,and PhWIND1 and PhSTEMIN1 were respectively controlled by 35 S promoters.The overexpression vector p G-PhWIND1 was genetically transformed into Arabidopsis thaliana and Petunia,and there was no significant phenotypic difference between Arabidopsis T0 positive plants and wild-type Arabidopsis thaliana.There was no significant difference between the obtained Petunia positive callus material and the wild-type Petunia leaf disc callus induced by hormones,but the Petunia positive callus material could not form adventitious buds normally in bud induction medium,but could form adventitious buds in hormone-free medium.It was speculated that overexpression of PhWIND1 could promote callus formation and inhibit the formation of adventitious buds in bud induction medium by influencing endogenous hormone synthesis.The superexpression vector p G-PhSTEMIN1 was transformed by Agrobacterium Tumefaciens to obtain transgenic positive materials from Petunia and tobacco.It was found that explants with overexpression of PhSTEMIN1 in Petunia and tobacco could directly produce adventitious buds at the explants’ wound without callus.It was speculated that PhSTEMIN1 could promote plant dedifferentiation and adventide bud formation.(5)Agrobacterium tumefaciens mediated genetic transformation of p G319 in Nicotiana alata,23 transgenic positive plants were identified through DNA detection,and three transgenic plants with different expression levels were screened out by real-time fluorescence quantitative analysis.It was found that overexpression of MIR319 caused serration of leaf edges and changes of ornamental characters of floral organs.The results showed that overexpression of MIR319 caused leaf edge serration,which resulted in the reverse roll of petals,the diameter of throat orifice and the length of corolla tube.At the same time,we also found that the plants with higher MIR319 expression decreased the anthocyanin content more significantly,indicating that MIR319 expression affected the synthesis and accumulation of anthocyanin in Nicotiana alata. |
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