| Potato is susceptible to abiotic stresses such as high and low temperatures,salt,ABA,cadmium and UV-B radiation.micro RNAs(mi RNAs)are a class of small non-coding single-stranded RNA molecules of 18-36 bp in length encoded by endogenous genes that degrade or repress the post-transcriptional translation process of their target genes through sequence complementation.mi RNA169 family is a widespread and relatively However,the mode of action of mi R169 in potato remains unclear.Different codons of the potato StmiR169 a isoform have the same mature sequence and whether there is phenotypic overlap or differential response in response to abiotic stresses.In this study,the promoters of eight members of the StmiR169 a isoform with the same precursor and mature body were constructed into GUS vectors,stably transformed into Arabidopsis thaliana and then observed for GUS staining of the isoform members.mi R169 mutants(STTM169/OE169a)were characterized in terms of their phenotypes in response to drought stress and regulation of growth and development,respectively,and a key gene was quantified by fluorescence.This thesis covers the following main aspects.1.Structural domain prediction of promoter sequences of eight StmiR169 a isoforms with the same maturity through the PLANTCARE website,which revealed that most promoters contained and abscisic acid response element ABRE,light response elements such as GATA-motif,Box 4,Gap-box,TCT-motif and hormone response elements including TGA and MYB,MYC,W-box,etc.2.The promoters of the eight StmiR169 a isoform members StmiR169a/b/c/d/e/f/g/h/mini / PBI121 were cloned and the plant binary expression vector PBI121 recombinant was constructed separately,and Arabidopsis thaliana was stably transformed using the inflorescence infestation method.The collected T0 generation seeds were sterilized with ethanol and sown in MS medium containing Kan and Cef(50 mg/ The T0 generation seeds were sterilized with ethanol and sown in MS medium containing Kan and Cef(50 mg/L)to screen for pure positive seeds of the T3 generation.The T3 generation histoculture seedlings at the 5-leaf stage were subjected to 0.1 μM ABA,20% PEG,150 n M Nacl,4°C treatment for 6 h.GUS staining analysis revealed that the StmiR169 a isoform could be divided into three subgroups based on tissue expression and stress response specificity,with StmiR169 a,StmiR169 c,and StmiR169 f as subgroup I.In StmiR169 b,StmiR169 g and StmiR169 h are subgroup II,with no expression in the hypocotyl but all other parts;StmiR169 d and StmiR169 e are subgroup III,with expression only in mature leaves and cotyledons.Both members of the StmiR169 a subtype were analyzed by fluorescence quantitative and semi-quantitative PCR under the same conditions,and both showed the same or similar expression patterns in terms of promoter activity intensity and tissue expression site specificity.3.Morphological comparison of the transgenic plants revealed that the STTM169 strain had sturdy stalks with a high number of stem angles and uprightness,while the overexpression plant(OE169)showed stem inversion.Analysis of the anatomical structure of the stalks by paraffin staining revealed an increase in the number of xylem,bast and middle ducts in STTM169,thus the change in vascular tissue was the main reason for the difference in stalk erectness between the transgenic materials.Further comparison of the vascular tissues of leaves and roots showed that the cell structure of the vascular cluster of STTM169 was dense,and the differences in the middle ducts between the different materials were obvious.4.Comparative analysis of net photosynthetic rate,stomatal conductance,intercellular carbon dioxide concentration and transpiration rate of potato STTM169 positive plants(S169)and overexpression plants(OE169)before and after drought stress at 35 days of seedling age revealed that the photosynthetic parameters of OE169 were significantly reduced and could hardly be restored to the control level after rehydration;although the photosynthetic parameters of S169 were lower under normal conditions than those of OE169 and Although the photosynthetic parameters of S169 were lower than those of OE169 and the wild type under normal conditions,they were significantly higher than those of the control in stress and after rehydration.Comparison of chlorophyll fluorescence parameters with DAB and NBT staining revealed that DAB and NBT deep staining appeared in concert with the rapid decrease in chlorophyll fluorescence parameters of OE169 under drought stress,suggesting that the decrease in OE169 photosynthetic system function caused by drought stress was accompanied by an imbalance of free radicals and that STTM169 was still able to maintain a higher photosynthetic system function under the same conditions,probably in association with the fact that STTM169 This may be related to the reduction of water evaporation under drought stress due to the appropriate reduction of leaf stomatal density.5.Tissue transparency staining was used to count the stomatal density,pore length,outer diameter width and inner diameter width of the upper and lower epidermis of the leaves of the transgenic plants,and it was found that the stomatal density of STTM169 was significantly lower than that of the control E3,and the stomatal apparatus became smaller,which,combined with the comparative results of photosynthetic parameters under stress,presumably enabled the lower stomatal density to reduce water loss under drought stress and maintain a higher cellular water potential The results of the comparison of photosynthetic parameters under stress led to the hypothesis that lower stomatal density could reduce water loss and maintain higher cellular water potential and photosynthetic system function.Transcriptome sequencing and quantitative fluorescence analysis also demonstrated that down-regulation of FAMA gene expression in STTM169(and up-regulation in OE169)was responsible for the stomatal differences between the two materials. |
