| Oil-tea Camellia is including important woody species which can product edible oil.Oil-tea Camellia is one of the most important woody oil species in China. The Camelliaoleifera Abel is planted in the most area and has the highest production. Until now, greatprogress were made on production, characters, post processing, oil nutrient content and oilstorage of Camellia oleifera Abel, However, the research on changes of fruits shape, theingredients in seeds and genes expressing of the important characters during development offruit and seeds were insufficient. Studying on the characters of fruits and seeds, thesynthesizing and storing law of the important nutritional ingredient during the development ofseeds can not only help us to promote the important cultivation measure of the Oil-tea Camellia,but also lay the foundation of improving the nutrition value by controlling the ingredients inOil-tea Camellia with molecular techniques. To seek the law of oil-tea Camellia fruits andseeds development, the changes of apparent traits, ingredients, genes expressions amongdifferent development stages of oil-tea Camellia fruits and seeds were studied, and thefollowing were the results.The fruits and seeds of Changlin No.4, Changlin No.40and Changlin No.166of Camelliaoleifera Abel were collected from May to October, and the changing rules of fruit shape, theweight of fruits and seeds, rate of seeds during the development of the fruits. The resultsshowed that most of the fruits shapes of the three varieties were round olive in May; andchanged greatly in August, and became the characters fruit shape of the variety until September.Fruit grew fastest from June to August, the weight, the diameter and the transverse diameterhad the greatest growing. After September, the weight and volume of the fruits kept constantly.The peel of fruits was changed slightly during the fruit development. The seeds grew continualbigger from June to October unsteadily. The fastest growing months were from June to August.Seed production percentage increased the fastest from June to August, too, and seeds kept steady in September. So it is concluded that the volume and weight of Camellia oleifera Abelfruits and seeds increase fastest during June and August.Ingredients of three Camellia oleifera Abel varieties seeds were measured from July toOctober. The results showed: The water content decreased gradually among seed developmentfrom May to October, especially from August to October. Oil content increased from July toOctober, especially from August to October. Changes of protein and starch were almost thesame with that of oil content. The soluble sugar showed little change in each month. The teasaponin content increased steadily with the mature of seeds. So it is concluded that theaccumulating stages of carbohydrate in Camellia oleifera Abel seeds were from August toOctober.RNA-Seq of Camellia oleifera Abel seeds from July to October were sequenced by usingsolexa technology. And in total,77050unigenes longer than200bp were got. There were99unigenes of13enzymes related to4fatty acid synthesis pathways,8unigenes of5enzymesrelated to fatty acid chain elongation,77unigenes of7unsaturated enzymes related tounsaturated fatty acid synthesis pathway and34unigenes of4important enzymes associatedwith squalene and saponin in steroid biosynthesis pathways. The gene expressions wereanalyzed during July and October according to RPKM values of each unigene.Relatively stable reference genes from6major species of oil-tea camellia were screened.According to reference genes of other species,15relatively stable genes which had stableexpressions in the seeds development were found out from RNA-Seq database. The stability ofthose genes which were expressed in roots, stems, leaves, flowers and seeds of Camelliaoleifera Abel., Camellia meiocarpa Hu, Camellia yuhsienensis Hu, Camellia chekiangoleosaHu, Camellia polyodonta F.C, How and Camellia semiserrata C.W. Chi were analyzed withqRT-PCR. The results showed that the most stable reference genes were different in differenttissues and species. But TUBα-3、CESA and ACT7α were relatively stable among all the tissuesand species. CESA was used to be reference gene. The expressions of29genes in4lipid metabolismwere analyzed by using qRT-PCR technology. And the relationship between expression ofgenes and their related metabolic prodction in different stages was studied. The expression ofthese genes by using qRT-PCR showed only a little difference compared with the result byusing RNA-Seq technique, such as BCCP, ECH and FAD8. The correlation between geneexpressions and variation of related metabolic production showed that expressions changes ofgenes in fatty acid synthesis, fatty acid chain elongation and unsaturated fatty acid synthesispathways were in advance to the changes of product contents. But the expression changes weresynchronous with that of metabolic production in the steroid biosynthesis pathways.Squalene synthase (SQS) gene and squalene epoxidase (SQE) gene were cloned fromCamellia oleifera Abel base on RACE technology. Full length of SQS was1490bp and thecoding sequence of SQS gene included2058bp, and encoding422amino acids. The full lengthof SQE genes was2058bp and the coding sequence of SQE gene included1605bp, encoding534amino acids. Then primers were designed according to the sequence of SQS and SQE genenucleotide sequence, and the SQS and SQE genes of Camellia meiocarpa Hu, Camelliayuhsienensis Hu, Camellia chekiangoleosa Hu, Camellia polyodonta F.C. How and Camelliasemiserrata C.W. Chi. were cloned using homology cloning technology. Relationship of SQSgenes and SQE genes of those six species were blasted. The result showed that the cDNAhomology of SQS and SQE in the six oil-tea Camellia species reached up to99.44%and99.47%respectively.The function of SQS and SQE was verified by pMDC32eukaryotic expression vector. Thegateway over-expression vector pMDC32of SQS and SQE gene in Camellia oleifera Abel wasconstructed. Then they were transformed to the Rhodotorula glutinis（Fres.）Harrison mediatedby agrobacterium EHA105. Then they were detected whether those transformed yeasts werepositive by PCR. Oil of yeasts was extracted after being cultivated, and the amount of squaleneand sterol in the oil were compared with those of wild type. The results showed that thesqualene and sterol content in oil of pMDC32-SQS Rhodotorula glutinis（Fres.）Harrison were reduced by26.26%and38.8%respectively compared with those in wild type. However, thesterol content in oil of the pMDC32-SQE Rhodotorula glutinis (Fres.) Harrison increased by202.62%, the amount of the squalene was reduced by41.09%. The result still showed that aftercultivated in the unit volume substrate in the same period, the production of the yeasts in thetwo kinds of transformed Rhodotorula glutinis (Fres.) Harrison increased by81.96%and183.52%respectively, and the total oil content increased by69%and175%respectively tothose of the wild type.Changes of morphology, ingredients and gene expression related to lipid metabolismduring development of Camellia oleifera Abel fruits and seeds were defined. and also made therelationship of genes expression and contents of their production clear accroding this paper,which provide a usful theory to find better cultivation methods and breed better varieties. |
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