Study On Floral Development And Gene Transformation Of Camellia Oleifera

Camellia oleifera Abel. is a typical flower and fruit co-development perennial evergreenplants. Blossom is an important basis for plant production and breeding. This paper analyzedthe physiological and biochemical variation during flower development of C. oleifera and mainfocused on the effect of environmental factors on the blossom. Based on the data results fromtranscriptome sequencing of flower, differentially expressed genes especially those related toflowering process were detected and the preliminary mechanism of C. oleifera flowering waselucidated. By RT-qPCR, the expression profiles of the key flower genes (FT and PI) wereanalysis and then the above genes were successfully cloned into the over-expressing and RNAinterfering vector. Moreover, gene transformation system was analysted and optimized. Theresults were listed as follows:1. During the flower differentiation, the content of soluble proteins gradually decreased in oldleaves, increased in stems. The content of soluble sugar were reduced in old leaves. Thesucrose content was sharply elevated in the stems and kept steady in leaves. The variation offructose content displayed similar change method in different organs of spring shoots.Comparing with the stem and old leaves, the content of fructose in young leaves was the mostabundant while was the least abundant in old leaves. In young leaves, the ratios of abscisic acidand auxin kept constant while in the stems and old leaves they varied largely.2. During floral formation, the contents of soluble protein and soluble sugar rised slowly indifferent parts of spring shoots. The content of sucrose and fructose varied dramatically, andthose were lower in flower buds than in leaves. The level of hormone in leaves showed atendency of “down-up-down”. The ratio of abscisic acid and gibberellic acid showed atendency of “up-down-up-down” in different parts of the spring shoot, The ratio of abscisicacid and auxin showed different tendencies in different parts of the spring shoot.3. During the period of flower differentiation, the contents of soluble protein and soluble sugar in the leaves were the highest in the short-day treatment. The content of sucrose was thehighest in the old leaves. The content of fructose in leaves was the highest in the nitrogentreatment and it was the lowest in the short day treatment. The content of auxin was lower instems than that in leaves. The content of auxin was highest in the heat treatment than that inothers treatments, and the lowest in light treatment. The content of gibberellin was the highestin new leaves under auxin treatment, and the lowest in stems of the control. The content ofcytokinin was the highest in young leaves under cytokinin treatment than that in othertreatmenyts. The relative gene expression of FCA in shoot tips was the highest under differentlight intensity treatments. The relative gene expression of FLC was the highest in seedling andthat of FT was the highest under the long-day treatment.4. The transcriptome sequencing obtained total of28,448,847reads and5,742,023,480bp. Thelength of N50was806bp. The number of unigenes longer than1kb was12,643. With24,534Unigenes being annotated in Swissprot,36,393in TrEMBL,36,400in Nr. The annotatedunigenes were then further classified into different processes and pathways by COG, GO andKEGG. RT-qPCR results showed that the expression levels of FLC, FCA and FT in the stemapex were lower than those of AP1, AP2, PI showed close relationship with the stamendevelopment. The digital sequencing of samples from six periods obtained a total of103,249,386reads. Alignment of the above data with transcriptome sequences. The analysisdetected26,861differentially expressed genes among the tested samples.5. The ORF of FT gene in C. oleifera was540pb that encoding179amino acids with themolecular weight was20KD and isoelectric point was7.74. The protein was most homologousto the FT gene in Actinidia chinensis Planch. The ORF of PI in C. oleifera was627bp andencoded208amino acids with the molecular weight of24KD and isoelectric point of9.11.That was most homologous to PI in soybean and sauteed green beans.6. MS medium was the most suitable medium for the regeneration of somatic embryosinduction and Changlin53variety was most easy for callus induction. TDZ,2.4-D areimportant factors influencing somatic induction. The combination of MS culture medium with 2mg/L IAA,0.5mg/L KT,0.5mg/L TDZ and500mg/L CH was most suitable to somaticinduction.7. Hygromycin concentration for callus selection pressure was120mg/L. The effect of explantpreculture time, Agrobacterium infection and co-culture time and AS concentration onHygromycin resistant callus was significant. Pre-culture time, infection time, co-culture timeand AS concentration had significant effect on the callus of pollution, and infection time andAS concentration had important effect on the pollution, secondly was the time of infection andpre-culture time. The conversion efficiency was the highest and the rate of pollution was thelowest under the condition of40days preculture,20min infection,5days co-culture and the200mg/L AS concentration.