| Longan（Dimocarpus longan Lour.）belonging to the Sapindus family is the evergreen tropical and subtropical woody fruit tree.Its embryonic development is closely related to the size and flavor of the longan fruit.The study of embryonic development of longan relies on the establishment of the embryonic system of longan.Somatic embryos and zygotic embryos are the most common embryonic systems.Under the natural condition,longan zygotic embryos are mostly heterozygous genotypes,genetic background analysis is difficult,and the formation of zygotic embryos depends on the development of the tree with long lifecycle.It is difficult to obtain continuous materials,which greatly restricts researches on the embryo development.The formation of somatic embryos is highly similar to that of zygotic embryos,and can be cultured in vitro with high purity,which is convenient for research and selection of materials and mechanism explanation at the cellular and molecular level.This article focuses on the formation of longan embryogenic callus（EC）to globular embryos（GE）,and set different temperature conditions（15℃,25℃,and 35℃）to explore the response of longan EC to temperature in the early stage of somatic embryo（SE）formation.We have constructed a transcriptome and s RNA database of longan EC in response to temperature changes,and obtained differentially expressed gene enrichment pathways which are alternative splicing and taurine metabolism.Subsequently,we also analyzed the gene family of the Dlo HSP（Heat Shock Protein）20 and the regulator Dlo SR of alternative spliceosome.Finally,we selected key SR genes with different expression patterns for cloning and transgene function.The main results are as follows:1.The morphology and the content of hormones,soluble sugar and Taurine in the early stage of SE formation of longan under different temperature conditionsThe thesis first analyzed and clarified the development process of longan EC to GE under normal temperature（25℃）using microscopic observation.Then the effects of different temperatures（15℃,25℃and35℃）on the development process of longan from EC to GE were analyzed,and the vitality of longan somatic embryos at different temperatures was determined by TTC method.The results showed that,compared with normal temperature（25℃）cultivation,relatively high temperature（35℃）and relatively low temperature（15℃）were not conducive to the formation of globular embryos.Under relatively high temperature,longan EC proliferates faster and can maintain a certain amount in a short period of time.The cell viability of longan was damaged for a long time;at relatively low temperature,the proliferation speed of longan was slow and the cell viability maintained well,but both of them were difficult to form a globular embryo.Enzyme-linked immunosorbent assay was used to analyze hormones including auxin（Indole-3-acetic Acid,IAA）,cytokinin（CTK）,and melatonin（Melatonin,MT）.The results showed that under the three temperature conditions,there was no change of the ratio of IAA and CTK.However,the hormone content in longan cells was different under different temperature conditions.Under relatively low temperature,the content of hormones IAA,CTK,and MT were higher than normal temperature.Relative high temperature did not change the content of IAA and CTK in longan cells.While the content of MT was significantly lower than normal temperature,which indicates that MT may be related to the formation of longan SE.In addition,the differential content of soluble sugar（SS）was noticed under different temperature conditions.Under relatively high or low temperature,the content of SS was significantly higher but the content of Taurine was significantly lower than that at room temperature,which indicates the formation of longan somatic cell globular embryos maybe related to the accumulation of SS and Taurine.2.Construction and analysis of a transcriptome and s RNA database in response to temperature changes in the early somatic embryogenesis of longanIn order to further explore the mechanism of early somatic embryogenesis of longan in response to temperature changes,longan cells at different temperatures were selected to construct libraries EC15,EC25and EC35.Each library includes three biological replicates,and the original data volume reached 6.5 G.The matching rate of the high-quality sequence to the genome was 73.27%-75.47%,and the Q30 value reached92.25%.A total of 27,260 gene were detected,of which 25,267 genes were known and 1,633 new genes were predicted.EC15 and EC35 had a total of 10,747 differentially expressed genes,of which 5466 were up-regulated and 5281 were down-regulated;EC25 and EC15 had a total of 3,696 differentially expressed genes,of which 1,639 were up-regulated and 2,057 were down-regulated;EC25 and EC35 had a total of 3,135differentially expressed genes,of which 1,644 were up-regulated,1,491down-regulated.The results of GO functional classification of differentially expressed genes were bioengineering,cellular components,and molecular functions,which contain 20,12,and 14 sub-branches,respectively.The GO enrichment was reflected in ribonucleoprotein synthesis,sodium proton transporter,and oligosaccharose transferase complex.KEGG functional analysis results showed that most of the differential genes were involved in carbohydrate metabolism,and the pathway with the highest enrichment coefficient was taurine and sulfinic acid metabolism.The metabolic pathway with the largest enrichment of differential genes was the alternative spliceosome,indicating that taurine acid and sulfinic acid metabolism and alternative splicing play an important role in regulating the growth and development of longan EC at different temperatures.A large number of Dlo HSP 20 were identified via the analysis of the transcriptome database EC15,EC25,and EC35.42 longan Dlo HSP 20gene family members detected in the transcriptome were analyzed for physical and chemical properties,subcellular localization,and phylogenetic evolution,gene structure,conserved motifs,promoter cis-acting elements,expression patterns as well as alternative splicing events.Most members have molecular weights between 15-30 k Da.Except for no distribution on chromosome 7,it is distributed on 14 other chromosomes.Each member contains an average of 62β-sheets,which is several times more than the number ofβ-sheets contained in the ideal ACD domain.Longan and the Arabidopsis HSP20 gene family were distantly related.It is speculated that the Dlo HSP20 gene has formed a relatively un-conserved gene family in the long-term evolution process.All member’s promoters contained a large number of cis-acting elements with unknown functions,most of the members have abundant stress response elements.The q RT-PCR results of all six Dlo HSP20 genes showed response to relatively high or low temperature treatments.In addition,the alternative splicing analysis of the HSP20 gene family found that A5SS events occur more commonly,and may have an important regulatory role in longan EC in response to temperature change.Post-transcriptional regulation is widespread in the process of plant somatic embryogenesis.In order to explore the post-transcriptional regulation of longan SE in response to temperature changes in the early stage,we also established s RNA databases using EC15,EC25 and EC35as materials.Each contains three biological replicates,and each database generates 23.35-25.13M raw data.After comparing with known s RNA databases,the ratio of the compared databases is between 91.46%and92.14%,and the sequencing data Q20 is as high as 99%.The analysis and comparison of these databases found that the identified s RNA types include mi RNA,si RNA,r RNA,sn RNA,sno RNA,t RNA,etc.,and the length of s RNA is mainly 24 nt.A total of 156 mi RNAs were identified with differential expression levels.After normalizing the expression level by TPM,it was found that mi R398b,mi R159a,mi R171f,mi R166a,mi R397a,mi R156a,mi R162a,and mi R408 were highly expressed.The differentially expressed mi RNAs were mi R170-5p,mi R162a,mi R168-5p,mi R408-5p and mi R398b,etc.The q RT-PCR analysis of the expression of pre-mi R398b and its target gene Dlo CSD1a during different temperature treatments showed that both of them responded to the temperature changes with different trends under high or low temperature conditions.While the expression pattern of pre-mi R398b and its target gene Dlo CSD1a were not completely opposite,indicating that different mechanisms may exist of Dlomi R398b in the response of high and low temperature conditions.3.Identification and analysis of longan alternative spliceosome regulator SR genesIn view of the fact that the analysis results of the transcriptome data of longan EC in response to temperature changes enriched the most alternative splicing bodies,we next carried out the longan genome-wide identification and expression analysis of the SR genes that regulates alternative splicing,and the results showed that there were 21 Dlo SR gene family members,which can be divided into 6 subfamilies RSZ,RS,SC,SCL,RS2Z,and SR,among which RS,RS2Z,and SCL subfamilies are unique to plants.Analyses of these 21 Dlo SR genes showed that the SR gene family of longan is relatively conservative in evolution,and their different subfamilies are both conservative and specific in the evolutionary process.Longan SR gene family contains multiple homeopathic elements related to growth and development,plant hormones,and stress.In addition,the analysis results of alternative splicing events indicate that different types of alternative splicing events play a different role in regulating the different stages of somatic embryogenesis.4.Cloning,expression analysis and functional verification of key differentially expressed SR genes in LonganThrough longan SR whole genome analysis,the SR gene family members Dlo RS43,Dlo SC18,Dlo RS2Z32,and Dlo-SR30 with different expression patterns were obtained.In order to conduct in-depth research on the functions of these genes,we obtained the full-length c DNA of the above SR genes through RACE.Successfully constructed p CAMBIA1301-35S-Dlo RS43-GUS,p CAMBIA1301-35S-Dlo SC18-GUS,p CAMBIA1301-35S-Dlo RS2Z32-GUSandp CAMBIA1301-35S-Dlo SR30-GUS overexpression vectors,and obtained transgenes after transformation of Arabidopsis thaliana by the flower dipping method.The expression pattern of longan SR gene was different under different light treatments.By analyzing the expression pattern of SR in EC under white light and blue light treatment,it is found that Dlo-SR33 is sensitive to blue light response.Different members of the longan SR gene have differential expressions in the same tissue and different tissues.Some SR genes showed tissue-specific expression pattern,indicating that the functions of different SR members in the growth and development of longan are different.The results of q RT-PCR of the SR gene of longan in the early stage of SE indicated that the SR gene have an important regulatory role in the stage of longan from ICp EC to GE.By measuring and analyzing the expression of Dlo RS2Z24,Dlo RS2Z32,Dlo SC18,Dlo SR30,Dlo SR33,and Dlo RS43 at different temperatures in the early somatic embryogenesis of longan,it is found that different SR genes respond to temperature changes in different modes.In general,longan cells respond stronger to low temperature.Specifically,under 15℃,the expression of Dlo SC18 and Dlo RS43 was higher than that at 25℃,while the expression of Dlo SR33 only increased on the 5th day and the other time points were lower than 25℃.The expression of Dlo RS2Z32and Dlo SR30 were not much different from 25℃,only the expression of Dlo RS2Z32 was lower than that at 25℃on the 1st day,The expression of Dlo SR30 showed a decrease on the 7th day.Under 35℃,the expression of Dlo RS2Z24,Dlo SR30 and Dlo RS43 showed different degrees of decline,while the expression of Dlo SR33 was higher than that of 25℃,and the expression of Dlo SC18 first decreased and then increased.The difference in Dlo SR genes response to high and low temperature patterns indicates different regulation mechanism in the development of longan EC.The GUS of Dlo RS43,Dlo SC18,Dlo RS2Z32 and Dlo SR30 transgene expressing plants was used to locate the SR protein in plants.The results showed that Dlo SC18 and Dlo SR30 can be colored on roots,stems,and leaves,but the degree of coloration in each part was different,Dlo RS2Z32 was mainly colored in the roots and stems,while Dlo RS43was mainly colored in the roots.Regardless of where it is,the expression level of Dlo SC18 and Dlo SR30 plants was much higher than that of Dlo RS2Z32 and Dlo RS43 plants in terms of the degree of coloration.Phenotype analysis of transgenic plants found that after overexpression of Dlo RS43,the growth of its roots was significantly inhibited,indicating that Dlo RS43 may play an important role in the development of plant roots.In summary,this study used methods such as physiological and biochemical,cytology,molecular biology and omics analysis,combined with the study of the model plant Arabidopsis thaliana,to comprehensively explain the response to different temperatures during the formation of longan somatic globular embryos.The results of this study provided theoretical guidance for clarifying the temperature response mechanism of longan cells and the improvement of the somatic embryo regeneration system,and laid a foundation for the development and flavor improvement of longan fruits as well as the biotechnology breeding of superior varieties. |
