Priliminary Functional Dissection Of Male-sterility Gene MSLP In Citrus

As the largest fruit crop, citrus are widely distributed in the world. Because of high nutritional value, medicinal value and health care value, citrus play an important part in our daily life. Seeds seriously affect fruit quality and economic value in citrus fresh market and processing, seedlessnesss has always been a goal of citrus breeding, our lab confirmed the seedless character of ‘Qianyang seedless’ Ponkan mandarin by cytology and genetics analysis compared with ‘Egan NO.1’. We employed SSH-c DNA libraries and microarry techniques and identified some differentially expressed genes between ‘Qianyang seedless’ and ‘Egan NO.1’. Meanwhile, the MSLP gene assosiated with ‘Qianyang seedless’ Ponkan mandarin seedlessness mechanism was cloned. In this study, the MSLP gene function was preliminarily dissected, the main results are as follows: 1. The subcellular localizationEnzyme digestion method was used to construct the subcellular localization vector PBI121-MSLP-GFP, MSLP and linked GFP gene expression were stared by Ca M35 s strong promoter, fluorescence of expression position determined by fluorescence microscope. In this research, we used onion as experiment material, Agrobacterium tumefaciens mediated genetic transformation method were applied to onion epidermal infection, the results showed that the onion epidermal nucleus has appeared fluorescence, suggests that MSLP gene located in the nucleus. 2. The yeast two-hybrid systemThe yeast two-hybrid system BD-MSLP fusion vectors were constructed, transcriptional activation and toxicity tests showed that the BD-MSLP fusion vectors were inactive. Mating method were applied to select the purpose sequence of the library, 5 genetic part information were found after sequencing and analysis of the multiple sequence alignment, 3 useful gene were obtained after matched with orange(citrus sinensis) genome database(http://citrus.hzau.edu.cn/orange/). Further analysis about the 3 gene expression protein sequence found as: DNA-binging storekeeper protein related transcriptional regulator; mitochondria-localized low molecular weight heat shock protein and one heat shock protein Dna J homolog protein. 3. Obtain positive transgentic tobacco and preliminary analyze tobacco phenotypeEnzyme digestion method was applied to construct overexpression vector p CAMBIA-1301-MSLP. In this research, Agrobacterium tumefaciens mediated leaf disc transformation has been used to importing p CAMBIA-1301-MSLP into tobacco plants. Induced buds and screening culture was done in medium which contained 2.5 mg/L hygromycin, buds were cutted when grow higher than 1 cm and putted into root training culture medium. Seedling planted into soil when grow up until blossom and yield seeds. Transgenic and wild type tobacco plants DNA were extracted, p CAMBIA-1301-MSLP plasmid used as positive control and wild type DNA as negative control, PCR testing to confirm that MSLP gene had been transferred into tobacco. Transgenic tobacco and positive control have gotten a bright aim strip at about 1300 bp by aim vector primer MSLP-F/R and about 500 bp by hygromycin primer Hyg-F/R while wild type had got nothing. The result of PCR testing indicated that there were 16 positive tobacco with aim gene. Preliminary analysis for transgenic phenotype found that there have no obvious differences in flower shape and pollen morphology compared with wild-type, but the NO. 3 tobacco imperfect pollen significantly higher than the wild type, the proportion imperfect pollen were 31.40%, the pollen viability test by carbol fuchsin dyeing showed that transgenic tobacco pollen vitality is normal. 4. Hongkong Kumquat RNAi interference experimentGateway technique was applied to construct the interference vector GRV-MSLP, GRV-MSLP carrier was transferred into Hongkong Kumquat while used Agrobacterium tumefaciens mediated epicotylar genetic transformation method. Induced buds and screening culture was done in MT induced buds medium which contained 500 mg/L Kan. The buds cultured under light after 2 weeks dark culture, and subculture for each 25 days. The resistant buds were cutted off with part of stem segments and cultured on MT elongation culture medium when the length grow above 0.5 cm, as subculture for each 25 days. When buds growth to appropriate size, cultured on the rooting medium to root induction. Two resistant seedlings have taken root, one of them is positive plantlet through PCR identification, more than 100 Hongkong Kumquat still in the rooting medium.