| Hickory, Carya cathayensis Sarg., is the Juglandaceae important woody oil plants. The hickory fruit is the famous dry fruit in the world, with high economic value, which is the important economic resource for farmers in the north-west of Zhejiang Province. However, the hickory grafting seedling is difficult to survive, the hickory variety is difficult to optimize, and the hickory quality is difficult to improve. Hence, study on hickory grafting propagation and molecular mechanism can provide new theoretical base for hickory variety cultivation. Rapid Amplification of cDNA Ends (RACE) technology was applied to clone plasma membrane intrinsic protein of hickory (CcPIP) using special primer of its EST fragment cloned by cDNA-AFLP technology. And Real time RT-PCR technology was applied to preliminary study its expression andcontrolling in hickory grafting seedling. The main results are summarized as following:1. The total RNA with high quality, integrality and no-impurity extracted with modified CTAB method was suitable to 3' RACE,5'RACE and Fluorescence Real Time RT-PCR analysis.2. The cDNA of hickory grafting seedling reversely transfered by using SMART TM PCR cDNA Synthesis Kit were integrate, and touch the requirement of experiment.3. The length of 3' sequence of CcPIP, amplified using 3' RACE System for Rapid Amplification of cDNA Ends Kit, is 450 bp, and its terminal has the poly A structure. The result indicated that it is the 3' end sequence of plasma membrane intrinsic protein using blast in the NCBI database.4. The length of 5' sequence of CcPIP, amplified using 5' RACE System for Rapid Amplification of cDNA Ends Kit, is 464 bp. The result indicated that it is the 5' end sequence of plasma membrane intrinsic protein using blast in the NCBI database.5. Using reversely transfered cDNA as template, we designed the special primers according to the cloned sequence and obtained CcPIP, the complete cDNA sequence in the hickory. The CcPIP is 99%, 99%, 98% and 98% identical to PIP of Vitis vinifera, Phaseolus vulgaris, Gossypium hirsutum, Petunia hybrida, respectively.6. Using ProtParam et al. bioinformation related software, we analized the cDNA of CcPIP. The molecular weight of cDNA is 75 860.6, the theoretical isoelectric point is 5.0, the instability index is 49.60, which show instability protein. The cDNA sequence includes 576bp open reading frame (ORF) which encodes 191 amino acids, among which there are, 8 acidic and 20 alkaline amino acids. 7.To determine the spacial-temporal expression pattern of CcPIP in hickory development, Real-time RT PCR techniques were applied. The results showed that CcPIP is expressed at low levels in the young bud, then the male inflorescence, the fruit, the young leaf and the stem, but strongly expressed in the young root system, which show identical function as water absorption. The expression trends in the scion are the same with that in the rootstock. The gene expressions were strongly induced both in rootstock and scion of hickory before grafting and declined sharply 3 days after grafting, which declined five times and two times. The expression in rootstock and scion were increased in the following days and reached to a high level 14 days after grafting, which was 7 times and 4 times stronger than that 3 days after grafting. CcPIP may be involved in the regulation of gene expression in water transportation of hickory grafting.8. The expression carrier of pCAM-1301-CcPIP was constructed using PGEM-T as middle carrier, which can be used for validating transgenic function in Arabidopsis.This paper studied the molecular function of CcPIP during hickory grafting process in the gene level. It will provide a basement for artificial regulation of hickory grafting using gene transforming, improving hickory grafting survive, and solving practical problem in hickory. |
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