Functional Analysis Of VvDRL1 Gene From Vitis Vinifera L.

NAC are plant-specific transcription factor,which shares a common feature,that the N-terminus is a highly conserved with a NAC domain containing 150 amino acids and the C-terminus is a highly variable transcription regulatory domain.Studies have shown that NAC transcription factors play an important role in plant flowering induction,embryonic development,the secondary wall thickening,cell division and so on.In this study,a NAC gene and its promoter,Vv DRL1 and PVv DRL1,were isolated from Vitis vinifera ‘Yatomi Rosa’ by homologous cloning method.The over expression vector p WR306-Vv DRL1 gene with 35 S promoter and recombinant expression vector p CAMBIA1391Z-PVvDRL1 with GUS gene were constructed.And these vectors were transformed into tobacco using the method of Agrobacterium through leaf disc transformation.Finally,stable transgenic plants were obtained.The results of this study are as follows:(1)transgenic plants grew slowly and the leaves were curled inwardThrough paraffin sections observation,it was found that the upper epidermis in main leaf veins of transgenic tobacco lacked 2-3 layers sclerenchyma cells and had significantly more space in the spongy tissue compared with the wild type.Based on the above study,it was inferred that Vv DRL1 gene played an important role in the regulation of plant morphological development.(2)the fruit of transgenic plant was small,and seed-setting ratio was lowThe experiments were operated to test the viability and fertility of pollen of transgenic plants by in vitro pollen germination method,I2-KI and FDA staining.The pollen germination rate of WT plant was 86.84% in 4-6 h by vitro culture method,but the transgenic plant was only 14.08%.Percent of pollen dyed to dark blue was 84.72% by 1% I2-KI staining,but the transgenic plant was only 21.95%,and the dyeing is lighter than the WT plant.Observed using a fluorescence microscope with a blue light,percent of the lighter pollen of WT was 92.45% by FDA staining,the transgenic plant was only25.00%.By GUS staining to the different development stage flower of transgenic tobacco containing PVvDRL1 gene,found this gene had the highest expression in stigma,the higher in pollen,and the dyeing is deeper following the longer of the development stage.The above results showed that VvDRL1 gene played an important role in the development of the flower.(3)the transgenic plants could delay senescenceUsing quantitative real-time PCR to YL,ML,ES and LS,it found that the expression of VvDRL1 gene was altered in leaves different periods,with the order: YL>ML>ES > LS.Physiological analyses of 8 weeks-old transgenic plants were performed,such as fresh weight,chlorophyll content,chlorophyll fluorescence and ion leakage.The results showed that chlorophyll content,chlorophyll fluorescence of transgenic plants leaves was higher than the WT leaves and ion leakage was lower than the WT.GUS staining was performed on the transgenic tobacco contained PVvDRL1 gene,found that the more younger the leaf was dyed deeper,and the more older was shallower.Results show that the VvDRL1 gene plays an important role in plant senescence.(4)Vv DRL1 gene was involved in leaf senescence by regulating ABAThe experiment treats the mature leaves of the grape using three hormones(IAA,6-BA,GA3)of promoting growth and four hormones(ETH,Me JA,SA,ABA)of promoting senescence,and analyses the expression of the Vv DRL1 gene through q RT-PCR.The result showed that the lower the expression is,the longer the time treating is,which showed that Vv DRL1 gene can control the senescence process of the plant by the four hormones such as ETH,Me JA,SA,ABA.Treated the mature leaves of tobacco with ABA,which found that the leaves of transgenic plants remain some green leaves,but the WT was none,and treated the mature leaves of tobacco with ETH,Me JA,SA,both the transgenic plants and the WT were all yellow.At the same time,assessing the expression of the key genes related to the ABA pathway,found that the expression was falling down.All these experiments above showed that the Vv DRL1 gene can delay plant’s senescence through ABA pathway.