| Loquat is perennial and evergreen plant belonging to the Eriobotrya of family Rosaceae,and it originates from South China.Main restricting factors for yield and quality are abundant and large seeds which lead to the edible parts of loquat are less.In addition,the efficiency of solar energy utilization of loquat is low and it is also easily inhibited by temperature variation that will affect blossoming and fruit bearing because of deseased photosynthetic capacity,especially cold stress in winter.PSII is vulnerable to temperature variation,especially in low temperature,which will leads to the more possible occurrence of photoinhibition of photosynthesis in plants.According to characteristic of PSII,a variety of response mechanisms are evolved to solve to photoinhibition situation,in which light harvesting proteins play a crucial role.Based on the former studies,tripioid loquat(GZ23×B431)with its parents diploid loquat Guizhou 23(GZ23)and tetraploid loquat(B431)were choosen as the research materials.Genes EjLhcb4.1,EjLhcb5 and EjLhcb6 encoded minor antenna proteins were cloned from triploid F1 loquats.Using bioinformatics methods,we analyzed the biological characteristics of the light harvesting proteins.Besides that,we examined the spatiotemporal expression pattern of these three genes in diploid,triploid and tetraploid loquats by q RT-PCR.p CAMBIA1300-EjLhcb4.1/5/6-GFP fusion protein were constructed and transiently expressed in N.benthamiana tobacco leaves to dected the subcellular localization.EjLhcb4.1/5/6 were subcloned in over-expression vector p FGC5941.These genes were transformed into col-0(wild type Arabidopsis thaliana)to observe phenotypic change of transgenic plants.Yeast hybrid vectors were constructed to observe the interaction between light harvesting protein and repair protein.In order to identify function and regulation mechanism of EjLhcb4.1/5/6 gene under low temperature few ways were used effectively.The major findings are as follows:1.Comparison of physiology activity among the different ploidy loquats under low temperature.The activity of triploid loquats photosynthetic enzymes and antioxidative enzymes and content of soluble sugar are higher than their diploid parent.Whereas there is no significant different between triploid and tetraploid loquats.With low temperature gradient treatment,the activity of antioxidative enzyme is higher below 0℃,but the activity of POD and APX enzyme exhibits a decreased tendency as temperature becomes lower.Rubisco enzyme expression is induced at low temperature.To detect relationship between accumulation of sugars and expression of photosynthetic related genes,high performance liquid-phase chromatography is performed to determine content of sucrose,fructose and glucose.There are significant differences in the total sugar content between control group and low temperature treatment group.The datas have reveal that the levels of soluble sugar are higher in low temperature treatment group.Decreased concentration of soluble sugars are determined along with the temperature down-regulation.Besides,the cold stress significantly enhances triploid loquats soluble sugar contents instead of the parent strains.Thus,it is suggested that there might be a regulation mechansim that mediate cold stress affects carbon fixation in plants but pattern of photosynthesis of triploid loquat are less vulnerable to low temperature.2.Bioinformatics analysis of EjLhcb4.1/5/6.Three genes are cloned from loquat,named as Ejlhcb4.1/5/6.The full ORF length of EjLhcb4.1 gene is 874 bp,encoding 290 amino acid residues,with a predicted molecular weight of 31.66 k D;The full ORF length of EjLhcb5 is 871 bp,encoding 289 amino acid residues,with a predicted molecular weight of 30.99 k D;Ejlhcb6 is 775 bp,encoding 290 amino acid residues,and has a predicted molecular weight of 37.39 k D.According to homologous comparison and evolutionary tree analysis,EjLhcb4.1 is most closely related to Qs Lhcb4.1 in Quercus suber and EjLhcb5 is tightly correlated with Pa Lhcb4.1 in Prunus avium,EjLhcb6 is closely connected with Md Lhcb4.1 in Malus domestica.EjLhcb5 and EjLhcb6 have evolved separately with Arabidopsis Thaliana.3.EjLhcb4.1/5/6 subcellular localization.The localization of EjLhcb4.1/5/6 in chloroplast are predicted by Bio-informatics system.p CAMBIA1300-35s-GFP-EjLhcb4.1/5/6 fusion proteins are constructed and transiently expressed in N.benthamiana tobacco.Fluorescence microscopy images show that the green fluorescence is coincide with the red autofluorescence of chloroplast,demonstrating that EjLhcb4.1/5 /6 locate in chloroplast.4.The interaction of repair protein Psb S and EjLhcb4.1,EjLhcb5 and EjLhcb6.p GADT7-EjLhcb4.1/5/6 and p GBKT7-Psb S vector are constructed to detect Ej Psb S inteact with EjLhcb4.1 by yeast-two hybrid technique.The research shows that Ej Psb S is inteact with EjLhcb4.1 in cold stress,minor antenna protein is therefore suggested to modulated protection for photosynthetic complex which Psbs involved.Although these studies not found significant inteaction between Psb S and EjLhcb5 and EjLhcb6,it still needs further verification.5.Function of Ejlhcb4.1/5/6 in transgenic Arabidopsis thaliana.p FGC5941-35S-EjLhcb4.1/5/6 fusion protein are constructed and transformed into col-0 and the transgenetic Arabidopsis thaliana grew well.When they were grown under low temperature treatment,wilt degree was much lower in transgenic lines than in wild type and transgenic lines can recovered to normal physiological morphology as soon as possible after back to normal environment.transgenic lines show higher survival rate and growth quantity.Additionally,all the transgentic Arabidposis thaliana exhibited higher Fv/Fm and electrical conductivity after chilling treatment.It also induced expression of light harvesting protein gene in the transgentic lines.These factors indicated that EjLhcb4.1/5/6 played important roles in maintaining the stability of plant photosynthetic system to avoid damage of PSII and mitigated the impact of photoinhibition for photosynthetic yield of loauats.In conclusion,EjLhcb4.1/5/6 can improve Arabidposis thaliana chilling tolerance and maintain its stability of photosynthesis at low temperature.However,the expression pattern and regulation mechanism in loquat with different ploidy need to be further studied. |
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