Study On The Mechanism Of Pigment Gland Morphogenesis And Correlation With Gossypol On Cotton

Cotton is not only the important fiber crop,but also is the largest protein and oil resouces following the soybean and rapeseed.In fact,cotton seed has not been utilized as a protein and rapeseed because of the presence of the gossypol which is taxic to humans and non-ruminant.Gossypol is a terpenoid aldehyde which is the important part of the prevention of pests and diseases.The plant will be hurted from pests and diseases and decline production due to the absence of gossypol.So it is important to study the mechanism of pigment gland formation and gossypol biosynthesis to cultivate new varieties with glandless seed /glanded plant.The near isogenic lines GI1（glanded seeds/ plants）and GI2（glandless seeds/ plants）were studied in this paper,The morphogenesis,density and diameter of pigment gland during the embryo formation and seed germation were observed under the microscope;The phenomenon of PCD was detected by the way of DAPI and TUNEL;The variety of different physiological and biochemical indexes during the pigment gland morphogenesis formation were measured;the content and degree of accumulation of gosspol in the process of pigment gland morphogenesis by high liquid chromatography（HPLC）and lead acetate sedimentation;The markers related to pigment gland by SRAP;The embryo of the key period from pigment gland were carried out to transcriptome seauencing by Seq-sequencing.The results were as following:1.The result of morphological showed that: The pigment gland primodium in GI1 was formed in the 25 DPA（25days post-anthesis）from flowering,The pigment gland cavity was formed in the 29 DPA from flowering,There was the occurrence of PCD during this stage;There were no pigment gland primodium,pigment gland cavity,and the occurrence of PCD in GI2.2.The physiological and biochemical studies during the process of the pigment gland showed that:（1）During the key period of the pigment gland formation（22 DPA、25 DPA、29 DPA）from GI1,The reduction of POD、SOD、CAT activity were resulting in the accumulation of H₂O₂,It is important for the PCD of the pigment gland.（2）During the period of seed germation,The activity of POD,SOD,CAT,APX,and the content of H₂O₂,ASA,carotenoid from GI1 were higher than GI2,The MDA content of GI1 was lower than GI2.This showed that the pigment gland can form during the germination process of seed from GI1,H₂O₂ can be produced in this process,the rising of H₂O₂ content can induce the rising of the antioxidant anzymes and antioxidant substances.3.The correlation study between the pigment gland and the gossypol showed that: the gossypol content of GI1 was higher than GI2;The gossypol was synthesized before the pigment gland formation,The gossypol was occurred in the pigment primodium,pigment cavity,and the intercellular space;There was no obvious correlation between the pigment gland and the gossypol content.4.The SRAP study about the pigment gland character showed that:（1）The segregation ration of pigment gland（glanded/glandless）was 3:1,the pigment gland density distribution was nomal distribution by the analysis the law of the segregation（glanded/glandless）and the density from pigment gland,This indicated that the character of the pigment gland（glanded/glandless）was belong to the quality trait which was controlled by a pair of genes,the number of the pigment gland was belong to the quantitative trait which was controlled by multiple genes.（2）The parents were amplified by 100 pairs primer combinations,The number of polymorphic primers were 33,The 33 pairs primer combinations were used to amplify between glanded DNA pool and glandless DNA pool,3 pairs primer combination were selected,the different fragment related the pigment gland trait was found.5.The transcriptiome study of the pigment gland formation showed that :（1）2.4×10⁸ raw reads were obtained from Y1（22DPA）,Y2（25DPA）,Y3（29DPA）,W（29DPA）,The sequencing number from every material were 6.6 G,7.84 G,7.42 G,6.88 G respectively.159699 transcripts and 95373 Unigenes were obtained by assembling,the total length of Unigene was 71902970 bp,The average length was 754 bp,N50 was 1239 bp.（2）By the Blast the Unigene with the public database Nr,Nt,Pfam,KOG/COG,Swiss-prot,KEGG,GO,49024 Unigenes were noted successfully.37743 Unigene had homologous information in Nr database,12143 Unigenes were noted in the 26 function categories from KOG database,29544 Unigenes were noted in GO database,11629 Unigenes were clustered 31 matabolic pathways in KEGG.（3）The blast about Unigene sequence was produced according to the order of Nr,Swiss—Prot,KEGG,COG database,the CDS and amino sequence from 38257 Unigenes were determined,the CDS and amino sequence from 17218 Unigenes were inferred by estscan.（4）570549 SNP loci were detected,60.6% belonged to the conversion type;50576 insertions were found,17515 SSR loci were found and 35769 pairs of primers were designed.（5）The expression of the different genes during the different period of the pigment gland from Y1,Y2,Y3,W were stastically analyzed,The number of differentially expressed genes were different in different combinations;The differentially expressed genes were divided into ten groups,and enriched the different function groups such as monolayer-surrounded lipid storage,lipid particle,external encapsulating structure,oxidoreductase activity,biosynthesis process,organelle part and so on;The key enzyme genes from the gossypol metabolic pathway,the phenylpropane metabolic pathway,and the PCD probably participate in the formation of the pigment gland.（6）Eight differentially expressed genes were selected for gene expression pattern analysis by q RT-PCR.The results showed that the expression trend of 8 genes in different combinations was basically consistent with the sequencing results,but the expression times of gene were different from the Sequencing results.