Drought Resistance And Expression Of Related Genes In Sugarcane Mediated With Ea-DREB2B Gene At Seedling Stage

Compared with the traditional breeding molecular breeding can greatly shorten the breeding cycle, and obtain the excellent characters of sugarcane growth.Drought responsive promoter rd29A was uesd to construct the highly efficient plant expression vector, and sugarcane clones mediated with Ea-DREB2B gene were confirmedbyparticle bombardment in Key Lab of Sugarcane Eco-Physiology& Genetic Improvement, Ministry of Agriculture. T7 and T11 were used as experimnet materials screened out by drought resistance evaluationwith strong drought resistance, acceptor matetial FN95-1702 and dominated cultivated varieties ROC22 were studied by contrast of originalvariety under artificial water stress to research the effects ofthe lethal days, physiological response and expression of Related Genes in transgenic sugarcane, and futher to explore the drought resistance and mechanism of transgenic sugarcane. Main research contents and results are as follows:1. The response of transgenic sugarcaneT7 and Tllto water stress in the lethal days,photosynthesis, osmotic adjustment, membrane damage and protective enzyme activity were better than the control, which showed that the transformation of foreign EaDREB2B gene enhanced drought tolerance of sugarcane.(1).The study of expression level of EaDREB2B gene was done by Real-time quantitative PCR, and the results showed the target gene could be detected in T7 and T11 but failed in FN95-1702. It is indicated that EaDREB2B gene was inherited stably in transgenic sugarcane.(2).Ea-DREB2B gene was highly expressed in leaves of T7 and T11under drought stress with the change characters of "increase-increase-decrease", and the expression was still 2 times than the normal water treatmentafter 10-days irrigation. T7 and Tl 1 kept high contents of chlorophylla, b and chlorophyll a+b content in leaves under drought stress.Compared to FN95-1702 and ROC22, T7 and T11 need more days todeath before the coming of critical point of waterunder continuous drought stress. It showed that the sugarcane mediated with Ea-DREB2B gene had stronger drought resistance.(3).The drought stress can lead to the rising of soluble protein, soluble sugar and proline content both in transgenic and control plant.The rate of rising intransgenic materials with Ea-DREB2Bgene was higher than in control plant. T7 and T11 had higher relative water content in leaves than FN95-1702 and ROC22. After irrigationtreatment, the content of proline and soluble sugar in leaves decreased sharply, soluble protein decreased slowly, and the relative water content of leaves increased.(4).The content of plasma membrane permeabilityand MDA inT7 and T11 with Ea-DREB2Bgene were lower than FN95-1702 and ROC22 under water stress, which indicated the damage degree of membrane lipid peroxidation was lower in transgenic sugarcane under drought stress, whera the protective enzymes(SOD,POD) activity of leaves intransgenic materials was higherthan control plant, which greatly reduced the degree of membrane lipid oxidation and the damage to the membrane.2. The drought stress resulted in overexpression of Ea-DREB2B gene in T7 and T11 leaves. Compared to normal condition, the expression of protective enzyme related genes (CAT, Fe/Mn-SOD, APX) of T7 and T11were higher than ROC22 and FN95-1702. It could be clearly seen thattransgenic sugarcanescavengingactive oxygen in cell and CAT gene expression was still high after re-watering.The expression of PRODH and SRM genes of osmotic adjustment substance were down-regulation under drought stress. The expression of PRODH gene was lower than FN95-1702, the expression of PRODH gene increased rapidly after re-watering; the overall trend of SRM gene expression was "falling-rise-rise". The expression of PP2C gene and ABF gene were consistent with the expression of Ea-DREB2B gene,the expression of PP2C gene and ABF gene were up-regulated under drought stress,the relative expression of transgenic sugarcane was significantly higher than FN95-1702.Ea-DREB2B transgenic sugarcane could effectively improve the drought resistance of sugarcane.