Cloning And Expression Vector Construction Of NfCCD7,a Strigolactone Synthesis Gene From Nervilia Fordii

Herba Nervilia Plicatae is one of the commonly used herbal medicines in Lingnan area that comes from Nervilia fordii（Hance）Schiltr.from Nervilia Genus of Orchidaceae family.It has the functions of moistening lung and relieving cough,clearing heat and cooling blood,dispersing blood stasis and detoxification,and is used clinically for the treatment of pulmonary diseases.The growth characteristics of Nervilia fordii are unique,with the corms as the asexual reproductive organ and its rigorous requirements of growing environment.Each plant grows only 1-2 new bulbs per year,with only one leaf per bulb.Since the low proliferation rate and high market demand,coupled with excessive excavation,the wild resources of Nervilia fordii are on the brink of extinction.However the artificial cultivation is far from meeting the market.In order to ensure the sustainable use of Nervilia fordii resources,it is imminent to find ways to expand its medicinal resources.In 2008,it was found that Strigolactones（SLs）have the function of inhibiting plant branching and play an important role in plant growth and development,and it has then been recognized as a new plant hormone with a terpene lactone structure.In the following years it has been shown that SLs also involved in promoting lateral root formation,inducing root hair elongation,regulating leaf senescence and mesocotyl elongation.Nowadays,it is known that SLs derives from carotenoids and the pathway from carotenoids to SLs requires at least four enzymes,the carotenoid isomerase D27,carotenoid cleavage dioxygenase 7,carotenoid cleavage dioxygenase 8 and cytochrome monoxygenase P450 family CYP711A1.Our group screened out candidate unigenes that might encode all reported genes related to SLs pathway by digging the previously obtained transcriptome data,indicating that the SLs pathway is very likely to participate in the regulation of the growth and development of Nervilia fordii.In view of the very few branching growth characteristics of Nervilia fordii,we will clone and explore the functions of related genes from the perspective of plant growth and development,and understand the regulation of the unique phenotypes of the plants of Nervilia fordii.Aim of the current thesis focues on cloning and preliminary analysis of the NfCCD7 gene.The main research contents include:（1）From the previous obtained transcriptome database of Nervilia fordii,we screened the Unigenes of synthetic genes related to SLs,including Unigene43499,Unigene63345 and Unigene64643,which were homologous with the MAX3 gene of Arabidopsis thaliana.On the basis of Unigene63345 sequence,RACE technique was employed to obtain the full length cDNA of the NfCCD7 gene.The coding region of NfCCD7 was analyzed by ORF Finder,and an 1836 bp open reading frame encoding a total of 611 amino acids was obtained.Protein domains of NfCCD7 were predicted and results showed that the conserved domains of NfCCD7 protein are mainly located between the 19th and the 608th amino acids,and belonged to the RPE65（Retinal pigment epithelial membrane recepter）superfamily.（2）Plasmids pRI 101 AN-EGFP and pRI 101 AN-NfCCD7/EGFP were constructed by the In-Fusion Cloning method using the pRI 101 AN-DNA as the expression vector,and the plasmids were transferred to the protoplasts of the tobacco leaves by PEG-mediated method.After cultured for a certain period of time,protoplasts were collected and observed under a fluorescence microscope.The expression of NfCCD7 in the chloroplast of tobacco cells was determined by comparing the subcellular expression of the two vectors in protoplasts of tobacco leaves.（3）The vector pRI 101 AN-NfCCD7 was constructed by means of In-Fusion Cloning using pRI 101 AN-DNA as an expression vector.After flora dipping transformation of Agrobacterium tumefaciens EHA105 and further steps,T₁generation transgenic Arabidopsis thaliana plants were screened,which laid the foundation for the functional characterisation of NfCCD7 in branching regulation of Nervilia fordii..