Identification Of Annexin Gene Family And Functional Analysis Of RsANN1a In Radish (Raphanus Sativus L.)

Radish(Raphanus sativus L.)is an important root vegetable crop originated in China.With its nutrient-rich and economic value,it is popular among people and widely cultivated in Asia and around the world.Radish is a cool season root vegetable crop,most of which are suitable for autumn and winter cultivation.High temperature is the main factor affecting the production and supply of radish in summer.Therefore,clarifying the genetic mechanism and cultivating excellent varieties with strong heat resistance have become an urgent problem to be solved.It is known that plant Annexin plays an important role in the normal growth and development of plants and participates in the entire life cycle of plants including embryo formation,seed germination,cork formation and cell cycle.Moreover,with the development of high-throughput sequencing technology and the advance of plant genome sequencing,the identification of annexin gene family was srported in several plant species.However,the sequence information about the annexin gene family is still unclear in radish.The annexin gene family has been shown to play an important role in plant heat-resistance in Arabidopsis,rice and lotus,but the related functions of the annexin gene have not been validated in radish.In this study,the annexin family genes were isolated and identified at the genome-wide level in radish,RT-qPCR was used to analyze the expression profiles of RsANN genes under five different treatments.With the floral dip method,RsANN1a gene and amiR-RsANN1a was transformed into Arabidopsis thaliana to verify its biological function effectively.The main findings were as follows:1.According to the published radish genome,10 RsANN genes were identified.RsANN proteins contained four typical Annexin domains and all of these 10 RsANN genes were located on the radish chromosome R3-R8.The analysis of protein characteristics showed that the number of amino acids of RsANN proteins was 314 to 324,and the relative molecular weight(MW)distribution was 35.57-37.06 kDa.The average instability coefficient(Ⅱ)of RsANN protein was 40.81(>40.00)indicating most of RsANN protein structures were stable.The prediction of fat index indicated that most RsANN proteins contained a large number of fatty amino acids,and the hydrophilicity analysis predicted that all RsANN proteins were predicted to be hydrophilic proteins.In addition,there were multiple post-translational modification sites in the RsANN proteins.Based on the published RNA-Seq data,it was found that the spatial-temporal expression profiles of RsANN family genes in different developmental stages and different tissues of radish.The results of RT-qPCR showed that the expression of 8 RsANN genes was significantly up-regulated after heat treatment(40℃)for 24h,especially RsANN1a,RsANN2a and RsANN4a.It was also found that except for RsANN1 a,the expression of other RsANN genes was induced by PEG treatment.Moreover,the expression of five RsANN genes showed significantly up-regulation compared with the untreated control under salt stress.The expression of all 10 RsANN genes showed significant up-regulation after spraying with 10 mM H2O2 solution.Furthermore,except for RsANN4b and RsANN8,the other 8 genes were up-regulated after treated by ABA.Overall,these results suggested that the RsANN genes might play an important role in response to various abiotic stresses.2.Using the advanced inbred line ’NAU-XBC’ as the experiment material,RsANN1a was isolated and the overexpression vector of RsANN1a gene and the amiRNA vector for silencing RsANN1a gene were constructed.The transgenic plants were identified by PCR at the molecular level and the results showed that RsANN1a and amiR-RsANN1a were successfully transformed into A.thalian.Subsequently,the 7-day-old wild-type(WT)A.thaliana and transgenic plants were treated at 45℃ for 2h,and then transferred to normal conditions for growth for 3-4d.The survival rate of WT line,RsANN1a OE and amiR-RsANN1a lines was 61.1%,88.9%,and 33.3%,respectively,indicating that the RsANN1a gene plays a crucial role in response to heat stress.These results confirmed the biological function of RsANN1a gene in response to heat stress,and would facilitate further dissecting the molecular mechanism underlying response to heat stress.in radish.