Cloning And Expression Analysis Of DoNI Genes In Dendrobium Officinale

Dendrobium officinale is a rare traditional, prennial and medicinal herb belonging to Dentrobium genus,Orchidoideae family. Polysaccharide is its main active conponent which is closely related with its multiple pharmacological functions such as strengthening the body’s immune functions, antitumor, anti-aging, anti-fatigue,and decreasing blood suger, etc. Currently it is also the major index for the quality judgement of D. officinale. Polysaccharide takes surose as its synthesis precursor, therefore it is correlated with sucrose synthesis tightly. Neutral / alkaline invertase(NI), which will irreversibly degrade sucrose into glucose and fructose to meet the hexose requirements of plant metabolism, may play an important role in plant sucrose metabolism and its regulation. The methods of homologous cloning, RT-PCR and RACE were applied in this study to clone the neutral / alkaline invertase gene in D. officinal, analyze its spatial and temporal expression in D. officinale plant, and study the effects of low temperature, p H and exogenous additives on its gene expression as well. The genetic transforming vector with NI gene was constructed and the heterologous transformation was carried out. The results revealed the preliminary function, expression and influence factors of NI gene in D. officinale which provide the theoretical basis for the metabolic regulation of polysaccharides and quality breeding of D. officinale. The main results of this paper are as follows:(1)Based on NI gene sequences of Elaeis guineensis in GenBank, RT-PCR and RACE methods, the full-length cDNA sequences of neutral / alkaline invertase were first cloned from D. officinale and was named DoNI(GenBank Accession No. KP742351) with a 2231 bp of sequence length, 1665 bp of open reading frame, encoding 554 amino acids, 62.8kD of relative molecular weight, isoelectric point of 6.56, and a typic neutral / alkaline invertase conserved domain. And DoNI shares a similar deduced amino acid sequences with that of Setaria italica, Oryza brachyantha. It also has a close relationship with monocots such as Elaeis guineensis, Phoenix dactylifera and Musa acuminata Colla which belong to the same clade.(2)By using real-time PCR, the temporal and spatial expression results of DoNI gene in D. officinale showed that it was expressed in roots, stems and leaves with the highest expression in stems. Its expressions in different growth periods of both tissue culture seedlings and field plants of D. officinale existed differences and the expression ranking from high to low was December > April>August which demonstrated the same variation trend with the polysaccharide accumulation.(3) Many factors may affect the DoNI gene expression in D. officinale. The optimal pH was 7.0 for its expression and overly acidic or alkali conditions could reduce its gene expression. Exogenous Ca2+ and Mn2+ treatments could significantly increase the expression quantity of DoNI gene and the effect of Mn2+ was better than that of Ca2+. Exogenous ABA treatment could greatly improve the DoNI gene expression, the expression quantity increased with the increase of ABA treating time and reached the peak at the treatment of 48 h. Increasing the sucrose concentration properly in the medium could also improve the DoNI gene expression in D. officinale. The gene expression was increased at the early stage of low-temperature treatment and decreased sharply with the heavier low-temperature stress and extension of treatment time which the value was much lower than the control. NaCl treatment could also promote the Do NI gene expression and the value reached the peak at the treatment of 24 h, then the expression quantity was decreased with the extension of treating time. Under adversity stress, D. officinale plants could enhance the catalytic abilty of sucrose degradation through increasing DoNI gene expression to meet the high energy requirements and synthesize compounds for coping with poor environments which indicates the gene may play a more important role in the adversity response of D. officinale.(4) The overexpression vector of DoNI gene was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation and12 positive plants were obtained finaly after Hyg selection which 9 of them were identified as the transgenic plants. The genetic transformation rate was 75%. The effects of DoNI gene on sucrose metabolism in Arabidopsis thaliana need further study.This study was first cloned the full-length cDNA sequences of Do NI from D. officinale and successfully obtained DoNI transgenic Arabidopsis thaliana. DoNI expression in different organs and growth stages was identical trends with the polysaccharide content.Low temperature and NaCl treatment was increased the expression of DoNI in a short time and it was similar to the NI gene in other plants. Ca2+, Mn2+, sucrose and ABA were obviously increased the expression of DoNI, indicating that these are common factors that influence the gene expression.