Function Of StInvInh1 In Potato Tuber Development

Cell wall invertase(CWI)irreversibly hydrolyzes sucrose into hexoses(glucose and fructose)extracellularly in plants,which are used for diverse metabolic and signaling pathways to regulate plant growth and development,and their activities in different crops determines the yield potential of crops by space or dose effect.Due to the glycosylation of the protein,the protein has the characteristics of stability.In addition to regulating its activity at the transcriptional and post-transcriptional levels,it is also an important way to regulate the activity of CWI at the protein level.Invertase inhibitor(INH)interacts with invertase and inhibits its activity,which is the main way to regulate CWI activity at the protein level.This regulatory approach can precisely regulate sucrose metabolism extracellularly,affecting plant growth and development and stress response.Our laboratory previously identified the potato(Solanum tuberosum L.)CWI suppressor StInvInh1 gene.Inhibition of StInvInh1 expression resulted in increased CWI activity and increased in vitro tuber size under heterotrophic conditions.The experimental results suggested that this gene has the potential to regulate potato tuber yield.On this basis,this study further observed the growth and development of transgenic lines under autotrophic conditions,and clarified the function of StInvInh1 gene in potato tuber development.At the same time,the transcriptional regulation function of StInvInh1 promoter was analyzed,and the potential mechanism of StInvInh1 regulation of tuber development was analyzed in combination with studies on transcriptomics,cytology,hormones and carbohydrate metabolism.The main results are as follows:1.Analysis of StInvInh1 promoter function and the acquisition of StInvInh1over-transgenic linesThe upstream 2.1 kb sequence of StInvInh1 was cloned as the promoter research object.Firstly,the promoter activity was identified by transient expression of double luciferase reporter system,and it was found that this sequence had promoter activity in tobacco leaves.Then,the expression vector of GUS driven by this promoter was constructed,and its stable genetic transformation strain in potato was obtained.In order to understand the temporal and spatial expression pattern of the promoter,the promoter activity was analyzed in 4-week-old tissue culture seedlings and test-tube potatoes at different development stages.The results showed that the promoter was mainly expressed in vascular bundles,and it was expressed in roots,stems,leaves and tuber organs.The expression level increased with the development of leaves,but decreased with the development of tubers.In addition,the promoter responds to hormones such as ABA,GA3,IAA and Me JA.The activity of the promoter studied above is similar to the expression pattern of StInvInh1 itself,which indicates that the promoter has the function of representing the expression pattern of StInvInh1 itself.In order to further clarify the effect of StInvInh1 on the development of potato plants and tubers,a StInvInh1 overexpression vector driven by StInvInh1 promoter was constructed,and 8over-transgenic lines were obtained through stable transformation of potato tubers mediated by Agrobacterium tumefaciens.2.Physiological function of StInvInh1 in potato growth and developmentPotato phenotype identification showed that inhibiting the CWI activity of StInvInh1 under greenhouse conditions could delay leaf senescence and increase the weight of single plant,the average weight of tubers,and the starch content of tubers.Similar results were obtained in field trials,suggesting that tuber swelling and dry matter accumulation mainly contributed to the improved tuber yield of the transgenic lines3.Molecular mechanism analysis of StInvInh1 regulating potato tuber developmentFor the potato development process,the invertase activity was higher in the stage of stolon to stolon expansion.However,with the expansion and development of tuber,the invertase activity decreased sharply to an undetected level.Inhibition of StInvInh1 increased CWI enzyme activity from stolon to stolon expansion stage,and maintained a higher level during tuber expansion development.Transcriptome analysis from stolon to stolon expansion showed that inhibiting StInvInh1 expression affected multiple pathways,including sucrose metabolism,auxin and cytokinin synthesis and signal transduction pathways.The results of hormone assay and tissue section showed that the content of IAA,CK and cell size of the transgenic lines were increased.These results suggest that silencing StInvInh1 increases and prolongs CWI activity during tuber development,resulting in enhanced and prolonged extracellular sucrose unloading,providing carbon metabolism and more material basis;In addition,signaling pathways such as IAA and CK are also involved in cell expansion and cell division,and they jointly regulate cell size.In conclusion,inhibition of StInvInh1 can release endogenous CWI activity in potato plants.By delaying leaf senescence,the supply of the "source" can be expanded,and the tuber yield can be improved by increasing the "sink" of tubers.Based on this,we propose a potential molecular model for inhibiting StInvInh1 to coordinate the "source-sink" relationship to promote tuber yield and quality.Our study provides new insights and genetic improvement approaches for fine-tuning CWI activity and regulating potato tuber development.