Cloning And Expressing Regulation Of Sucrose Non-fermenting-1-related Protein Kinase-1 (SnRK1) From Pingyitiancha (Malus Hupehensis Rehd.) And Researching Of Its Function

SnRK1 complex plays a key role in carbon and nitrogen metabolisms of modle plants. Under water culture condition, we have cloned two subunits of SnRK1 complex from pingyitiancha seedlings. The aim of this study was to obtain a better insight into the character of the SnRK1 complex subunits encoding genes in respond to environmental signals and in carbon metabolisms in fruit trees. The expression patterns of MhSnRK1 and MhAKINβγin different tissues at different conditions were analyzed by quantitative real-time PCR. The MhSnRK1 gene derived from pingyitiancha was heterologously expressed in tomato plants to study the biological function of the SnRK1.1. In an attempt to isolate MhSnRK1 gene, two EST sequences were used to design GSPs for 5′-RACE and 3′-RACE isolation of MhSnRK1; one EST sequence was 433-bp (CN445648) and the other was 466-bp (CX024405). Analysis revealed a 2063 bp sequence obtained by RACE was the full-length cDNA of the MhSnRK1 (Malus hupehensis Rehd. SnRK1, EF690362) gene. The nucleotide sequence of MhSnRK1 contains a 166-bp 5'-untranslated region, a 1548-bp open reading frame encoding a protein of 515 amino acids with a mass of 58.7 kDa, and a 348-bp 3'-untranslated region. The deduced amino acid sequence of MhSnRK1 showed 82.34, 87.57, 86.02, 84.08 and 82.14% identity to the Arabidopsis AKIN10, tomato LeSNF1, potato StSNF1, tobacco NPK5 and Pea, but it exhibited only 66.67 and 66.22% identity to the Barley BKIN12 and Rye RKIN1. The phylogenetic tree showed MhSnRK1 belongs to the type 1 (SnRK1) subfamily of the snf1-related kinases (exemplified by Arabidopsis AKIN10) rather than the SnRK2 subfamily (exemplified by Arabidopsis ASK1) or the SnRK3 subfamily (exemplified by Wheat WPK4). We also produce a partialβγ-subunit (MhAKINβγ) with 3′-RACE. After a 100-fold dilution of the first PCR product, the product was used for a second PCR with the primer of SN32, from which a 3′-RACE 1112-bp fragment was obtained. Analysis of this fragment showed its nucleotide sequence was highly conserved with those in other plant species (including fragments of apple, maize and Medicago truncatula) Therefore, this fragment was considered a pingyitiancha AKINβγhomologue and was designated as MhAKINβγ.2. Expression of MhSnRK1 and MhAKINβγgenes was studied in leaves, stems and roots of two-week-old pingyitiancha seedlings by quantitative real-time PCR analysis using subunit-specific probes. An r18S gene was used as an internal standard. The results revealed that transcripts of MhSnRK1 and MhAKINβγwere detected in all tissues analysed, but the two genes expression differed among root, stem and leaf tissues. Higher levels of MhSnRK1 transcripts were expressed in leaf tissue relative to the root and stem. Similar expression patterns were observed from MhAKINβγ, with the highest transcriptional level also occurring in leaf tissue. To understand the short-term effects of nitrogen and osmotic stress on expression of MhSnRK1 or MhAKINβγ, transcriptional levels in different plant tissues were monitored by quantitative real-time PCR analysis during induction by NO3- and PEG water stress. Control plants treated with KCL or grown in normal nutrients exhibited nearly consistent levels of MhSnRK1 or MhAKINβγfrom 0 to 48 h. The NO₃^- does not affect MhSnRK1 expression as early in leaf, yet it inhibits MhSnRK1 expression after 48 h. Meanwhile, NO3- feeding reduces MhSnRK1 expression 5-fold in root within 24 h, but the transcript levels raise to control levels after 48 h. Under PEG water stress, MhSnRK1 mRNA levels increased in roots within 24 h but then declined to the levels of control after 48h. In leaves, MhSnRK1 expression decreased as early, and then increased after 48 h. Interestingly, MhAKINβγexhibits a similar expression pattern as MhSnRK1 during the NO₃^- and PEG treatment. When KNO₃ was resupplied, pMhAKINβγtranscripts decrease 3-fold in root within 24 h, and low in leaf after 48 h compared with the control. A 13-fold induction of pMhAKINβγexpression was detected after PEG treatment for 24 h in root, and then the transcript levels declined to the levels of control after 48h. MhAKINβγtranscripts in leaf decreased 10-fold within 24h compared with the control . 3. In order to determine the function of MhSnRK1 in growth and development of plants cell, a construct containing full-length cDNA of MhSnRK1 gene in sense and atisense orientation driven by the constitutivecauliflower mosaic virus 35S promoter was assembled and introduced into tomato. The quality of the transgenic tomato fruits was also analysis. Compared with the wild-type, the soluble sugar and starch levels of the transgenic plant fruit were increased by approximately 20%-30% and 44%-56%, respectively. Organic acid and total soluble protein were 44%-88% and 55%-76% of the control concentrations, respectively. The results indicate MhSnRK1 has obvious effects on the quality of the tomato. Our research also showed that the fruit diameter of the transgenic plants was much thicker at the early stages of fruit development than the wild type, but were similar at later stages; the transgenic fruit ripened ten days earlier than wild type fruit.The starch content of the leaf from transformants was assayed. we found that the starch content in transgenic lines was higher than wide type in leaf at the end of the day, for example, the starch content in T2-5 transgenic line was increased by 105% compared with that in the wide-type.