| Jinjiang oyster（Crassostrea ariakensis）is an important marine shellfish in my country,which has important economic value and ecological function.Glycogen is the main form of carbohydrate storage in animals.It is the most direct and effective energy storage substance in oysters and plays a vital role in the physiological metabolism of oysters.As an important quality trait of oysters,glycogen content directly affects the flavor and nutritional quality of oysters,and is an important criterion for judging oyster meat quality traits.At present,in response to the requirements of oyster genetic breeding and oyster bioinformatics analysis,a method for determining glycogen content in oysters that is efficient,simple,accurate and reliable,and with low cost has not been developed and reported.Different geographical groups and different developmental stages of C.ariakensis.There is no systematic study on the changes of individual glycogen content and metabolic regulation mechanisms.In this study,based on the collection and analysis of the morphological characteristics of five geographic populations of C.ariakensis in Shandong Binzhou,Shandong Dongying,Shanghai Luchaogang,Guangdong Guangzhou,and Guangxi Beihai,a trace reaction system was constructed based on the principle of anthrone colorimetry for the determination of sugar.Based on this method,the seasonal variation of glycogen content in different tissues of Dongying C.ariakensis,Shandong Province and its relationship with the development of gonads were analyzed based on this method,and based on transcriptomics data,the glycogen metabolism-related factors were obtained and preliminary analyzed.Glycogen synthase gene and glycogen phosphorylase gene.This research mainly includes the following four aspects of research content:（1）Collection and morphological analysis of different geographic groupsA total of 305 individuals were collected from 5 individuals of the inshore oyster group in Binzhou,Shandong,Dongying,Shanghai,Guangzhou,Guangdong,and Beihai,Guangxi.After species identification,the shell height,shell length,shell width,body weight,shell weight and other data were measured.Use SPSS 22 to analyze the correlation between the morphological data of C.ariakensis.The results show that the correlation coefficient between body weight and shell weight is the largest;the correlation coefficient between shell height and shell length is the smallest;the correlation between the measurable traits of Guangxi Beihai population is quite different,and there is no significant correlation between shell height and shell width,body weight and shell weight.The weight and shell weight of Binzhou and Dongying populations are significantly correlated with shell height,shell length and shell width traits;the morphological traits of Binzhou population are not significantly correlated;the morphological traits of Dongying population are only significantly correlated with shell length and shell width.The correlation between the measurable traits of the Shanghai population was extremely significant（P<0.01）,and the correlation coefficient was between 0.492 and 0.996.In summary,the correlation between shell length,shell width,and shell height shows that compared with the other four groups,the shell type of the Shanghai C.ariakensis population is more uniform,and the shell types of the other four C.ariakensis populations are more abundant.（2）Establishment,optimization and application of the micro-reaction system and method for the determination of glycogen contentUsing fresh C.ariakensis soft tissue as the experimental material,the optimal reaction conditions were determined by comparing and analyzing the absorbance values of different anthrone-sulfuric acid ratios and different reaction times,and the lowest detection limit,repeatability,stability and accuracy of the method were evaluated.Establish a reaction system for the determination of trace glycogen.The results of the study are as follows:The volume of the optimized micro-reaction system is 300μL,which mainly includes 200μL of 0.2%anthrone sulfuric acid solution and 100μL of the sample solution;the reaction time of the boiling water bath is 10 min;the minimum detection limit of glucose of this method is 0.001 5 mg·m L-1,the coefficient of variation of the standard curve is less than 4%,the detection sensitivity is high,and the repeatability is good;after the color reaction is completed,the absorbance value does not change within120 minutes at room temperature,and the stability is high;The recovery rate of standard addition for mantle,gill,lip,gonad,hepatopancreas,and adductor muscle is between 95.3%and 105.8%,which is highly accurate.The micro-reaction system established in this study has the advantages of small amount of reagents,simple operation,and low cost of a single sample.At the same time,it has high repeatability,stability,and accuracy,and is suitable for batch determination of large quantities of samples.Using this method to determine the glycogen content in different tissues of C.ariakensis showed that the four tissues of mantle,gonad,lip and hepatopancreas had higher glycogen content.Among them,the glycogen content of the lip was the highest,which was 51.98%of dry weight,followed by The gonad is 47.28%of the dry weight.The tissue with the lowest glycogen content is the adductor muscle,which is only 4.67%.In different individuals,the glycogen content of adductor muscle is not significantly different,between 4.1%and 5.4%.（3）Annual change of glycogen content in Dongying population in Shandong and its relationship with gonadal developmentThe glycogen content of six tissues including adductor muscle,gills,mantle,hepatopancreas,lips,and gonads of individuals in Dongying population from May 2019to April 2020 was determined.The gonad development was staged based on tissue sections and the sugar was analyzed.The relationship between the original change rule and the characteristics of gonadal development.The results are as follows:combined with the slice results,the near-gonadal development of the Dongying oyster population is divided into six periods:formation period,proliferation period,maturity period,discharge period,and exhaustion period;Dongying oyster in Shandong has entered the formation period successively from March to April.Most individuals are in the proliferation period.May and June are mainly individuals in the mature period,and the concentrated discharge period mainly occurs in July and August.In September and October,with the discharge of sperm and eggs,the number of individuals entering the depletion period gradually increased,and in November they all entered the resting period.During the formation period,the glycogen content of each tissue is high.As the gonadal development enters the proliferation and maturity phase,with the maturation of the gametes,the glycogen content of the gonadal tissue decreases most significantly,from 581.6 mg·g-1 to 202.8mg·g-1,other organizations also show similar trends.With the discharge period when the mature period comes,the glycogen content of the gonad tissue increases slightly,and the gamete enters the resting period after the discharge is completed,and the glycogen content does not change significantly.Among the six tissues,the glycogen content of adductor muscle has a small fluctuation range.The glycogen content is the lowest in the discharge phase,which is 43.9 mg·g-1,and the glycogen content is the highest in the formation phase,which is 72.5 mg·g-1.（4）Screening and analysis of two genes related to glycogen metabolismRelying on the Pac Bio third-generation sequencing platform to carry out three-generation full-length transcriptome sequencing,screening for genes related to glycogen metabolism,and bioinformatics analysis of genes.The results showed that 347,212original CCS（circular consensus）sequences were obtained,and 277,460 full-length non-chimeric sequences were screened out（accounting for 79.91%）;the full-length non-chimeric transcripts after clustering were performed with non-full-length transcripts Sequence correction,80,813 high-quality full-length transcripts were finally obtained,accounting for 93.91%of the total consensus sequence;after cluster correction,redundant sequences in high-quality transcripts were removed,and 48,781 non-redundant transcripts were finally obtained Sequence;Gene structure annotation identified a total of7 repeat types of microsatellites,and predicted 1928 variable splicing events,30,724complete ORFs,8530 Lnc RNAs,and 1,350 transcription factors.Multiple databases were used to annotate transcripts.A total of 29,550 transcripts were annotated in the GO database,and 19,727 annotated transcripts were annotated in the KEGG database.Root screening obtained the full-length sequences of Glycogen synthase and Glycogen phosphatase genes in the glycogen metabolism pathway.The second-generation transcriptome analysis showed that the expression of glycogen synthase and glycogen phosphorylase genes reached the lowest level in the mature stage of the gonadal tissue,and the change trend of the expression of the two genes was consistent with the change trend of glycogen content in different periods. |
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