Studies On The Mechanism Of The Transgenic Plants Dwarfing Caused By Overexpression Of Heat Shock Transcription Factor SlHSFC1 In Tomato

Tomato(Solanum lycopersicum L.)contains important nutrients essential to human health,such as lycopene,ascorbic acid(vitamin C)and vitamin A(β-carotene).Lycopene has antioxidant properties and is believed to prevent cancer or cardiovascular disease.In the process of agricultural production,growth and development are the main factors affecting tomato yield.Therefore,understanding the regulation mechanism of tomato growth and development plays a very important role in improving tomato cultivation techniques,cultivating excellent new varieties,and increasing tomato yield and quality.Heat stress transcription factor(HSF)not only participates in the response of plants to various stresses,but also participates in the regulation of plant growth and development.HSF is a large multi-gene family,which can be classified into A,B,and C.Among them,A has heat-shock transcription activity,and C plays an inhibitory and regulatory role,but the function of C is still unclear.This study cloned the heat shock transcription factor C1 gene(SlHSFC1),analyzed the bioinformatics characteristics of the gene,analyzed its expression pattern in response to hormones such as GA,and analyzed that the overexpression of this gene in tomato leads to dwarfing of the T1 generation plants The reason for this gene overexpression was analyzed,and the influence of the gene overexpression on the GA content and the expression of related genes in the GA synthesis and signal transduction pathways was analyzed,in order to analyze the physiological and molecular mechanisms of tomato plant dwarfing caused by the gene overexpression,in order to clarify the gene The researches on biological functions and molecular mechanisms of tomato growth and development have laid the foundation.The results of the study are as follows:(1)Expression characteristics and sequence analysis of SlHSFC1 geneUsing the g DNA of wild-type tomato AC++ as a template,a full-length 1127 bp sequence of tomato SlHSFC1 gene was amplified by the high-fidelity enzyme Prime STAR.Sequence analysis showed that the SlHSFC1 gene encodes a protein of369 amino acids with a molecular weight of 41.1k Da,a theoretical isoelectric point p I of 6.05,and an instability index of 58.43,which is an unstable protein.The secondary structure of SlHSFC1 protein was predicted by software SOPMA,and it was found thatα helix accounted for 28.46%,β turn accounted for 6.23%,extended chain accounted for 13.01%,and random coil accounted for 52.30%.Real-time fluorescence quantitative PCR analysis of the expression pattern of the SlHSFC1 gene showed that the SlHSFC1 gene all responded under the treatment of GA,SA,Eth,JA hormones and photoperiod.(2)Obtained SlHSFC1 overexpression and CRISPR-Cas9 knockout transgenic plants The overexpression of tomato SlHSFC1 gene was constructed,and tomato explants were genetically transformed by Agrobacterium-mediated transformation.SlHSFC1 overexpression and knockout transgenic plants were obtained,and 5 overexpression lines were identified by q RT-PCR;The T1 generation plants of these transgenic plants are all dwarfed,and the development of roots and hypocotyls is blocked.At the same time,a CRISPR-Cas9 knockout vector of this gene was constructed,and two SlHSFC1 knockout mutants were obtained.The T0 generation plants have normal morphology.(3)The dwarfing traits of the SlHSFC1 overexpression transgenic tomato plants can be partially restored by exogenous GA3SlHSFC1 overexpression transgenic plants showed dwarfing in the T1 generation.Treatment of SlHSFC1 overexpression transgenic plants and wild-type seedlings with GA3 showed that GA3 can only partially restore the growth of dwarfed transgenic seedlings,but it is difficult to restore to normal form.It is speculated that SlHSFC1 may affect the synthesis of GAs or signal transduction pathways..(4)SlHSFC1 overexpression did not inhibit GA synthesis of dwarf plants but hindered GA signal transductionThe results of gibberellin content determination showed that there was no significant difference in the GA1,GA3 and GA7 content between the SlHSFC1 overexpression transgenic tomato dwarf plant and the wild-type plant,and the overexpression of this gene resulted in a significant accumulation of GA4;therefore,the overexpression of SlHSFC1 was not inhibited The GAs synthesis of dwarf plants,on the contrary,promotes the increase of gibberellin GA4.The results of q RT-PCR analysis showed that the overexpression of SlHSFC1 can increase or inhibit the expression of related genes in the gibberellin synthesis pathway of transgenic tomato dwarf plants,confirming that there is no overall inhibition of GAs synthesis at the gene expression level.However,the expression level of GAs receptor protein gene GID1 was significantly down-regulated,indicating that SlHSFC1 may negatively regulate the signal transduction pathway of GAs to cause plant dwarf and affect plant growth and development.In summary,the mechanism of overexpression of SlHSFC1 leading to dwarfing of transgenic tomato plants is not to inhibit GA synthesis,but to down-regulate the expression of DELLA,especially the GID1 gene,which hinders GA signal transduction and ultimately leads to dwarfing of transgenic plants.