| Hot pepper (Capsicum annuum L.), a member of Solanaceae family, is growingabundant in tropical, subtropical and temperate regions. Nowadays human demands increasedon pepper due to further requirements i.e. food processing industry and medicinal purposes(United Nations Food and Agriculture Organization statistics). Therefore, recent researchesfocus on germ plasm and biotechnology breeding of pepper. Furthermore, modern technologyand conventional plant breeding have been used to improve the pepper tolerance to differenttypes of stress. In pepper, one of tonoplast intrinsic protein gene (named CaTIP1-1) wasinvestigated using cDNA-AFLP method. In the current study, the overexpression ofCaTIP1-1gene decreased chilling tolerance in transgenic tobacco plants; on the other hand, itincreased the tolerance to osmotic stresses. Also, another plasma intrinsic protein gene ofCaPIP1-1was isolated according to transcriptome databases of Phytophthora capsiciinoculation. This CaPIP1-1gene decreased the tolerance to osmotic stresses in silencedpepper. It is possible that studying the aquaporins leads to narrow the gaps between pepperand other crops of Solanaceae family. Therefore, in this study aquaporin genes wereidentified from pepper genome sequencing database using the phylogenetic analysis. Apepper regeneration system was also improved in three varieties. And the main results in allexperiments were as follow:1. Seed sequence of PF00230was used in CM334protein database (Protein codinggenes for the pepper V1.55) with the HMMER3.0software. At last56AQPs in pepper weremapped according to genome sequence. Their distribution on chromosomes greatly variedfrom1gene in chromosome05and07,2genes in chromosome09and12,3genes inchromosome04,5genes in chromosome02,08and10,6genes in chromosome03,9genesin chromosome06and11genes in chromosome01. The number of introns in each aquaporingene varied from zero intron in CaPIP2-7, CaTIP1-11, CaTIP1-12, CaTIP1-13, CaTIP1-16,CaSIP2-1into a maximum of four introns in CaNIP1-1, CaNIP3-1, CaNIP3-2, CaNIP4-2,CaNIP6-1. Interestingly, all the introns exhibited CT/AC or GT/AG splice junction’s model. Only one splice junction’s model was showed in the same gene. Forty two aquaporins,containing highly conserved dual Asn-Pro-Ala (NPA) motifs (or NPA-like motifs), wereclustered into five distinct subfamilies; TIPs (23), PIPs (15), NIPs (10), SIPs (3) and XIPs (2).2. The expression of27aquaporin gens was detected in the24tissues using RNA-seqdatabase (21AQPs were identified in the different subfamily and6AQP genes were notdetected in the Pepper Genome Annotation v.1.55). Some of aquaporins are involved in theregulation of pepper fruit development and others may be related to the pepper fruit ripening.The expression of CaSIP1-2, CaTIP1-1and CaPIP2-9gave high level in different tissues. Onthe contrary, the expression of CaPIP2-9was lower at the stage of CMPCB10, CMPLB10and ECPLB10. While, the expression of CaNIP7-1, CaNIP4-1, CaNIP3-1, CaNIP1-3,CaNIP4-3, CaNIP3-2, CaPIP1-5, CaTIP1-13, CaTIP1-10, CA04g3870and CA00g98340waslower in all the stages. The expression of CaNIP4-1, CaNIP7-1, CaNIP6-1, CaTIP3-1,CA10g07870, CA00g57100and CaTIP1-1(not in ECW30R) were upregulated during thefruit development stages. While the expression of CaSIP1-2, CaPIP1-4, CaNIP4-2,CaPIP2-5, CaPIP2-8, CaPIP2-9, CaTIP1-1, CaTIP3-2, CaNIP5-1and CA08g13100weredownregulated during fruit development stages. Increasing the amounts of CaNIP1-1,CaNIP3-1, CaTIP3-1and CA00g98340transcripts and decreasing amounts of CaNIP1-3,CaNIP6-1, CaTIP1-1, CaPIP2-8, CaPIP2-9transcripts both can be detected after breakerstage.3. The CaTIP1-1localized to the tonoplasts by transient expression. CaTIP1-1contained one intron of1664bp in length with GT/AG splice junctions. The tissue expressionpattern was higher transcript in all of flowers, immature fruits and young seeds than roots,stems and leaves. The pepper plants with silenced CaTIP1-1in P70showed growth inhibition.Expression of the CaTIP1-1was downreglated by using50μM HgCl2and100μM fluridone(inhibitor of ABA biosynthesis) and also under chilling stress conditions. With less watersupply, CaTIP1-1transcripts could be increased in case of using0.15M NaCl,0.15Mmannitol,5mM salicylic acid and100μM abscisic acid.4. Overexpression of CaTIP1-1pepper gene in tobacco decreased the chilling toleranceand recovered slowly after exposing to low temperature stress. Transgenic tobacco plantsincreased the stomatal aperture opening after incubation with stomatal colsure solution andapproximately86.3%stomatal aperture was still opened at0.31±0.047. The photosyntheticrates of tobacco wild type seedlings were inhibited45.29%after3d of exposure to lowtemperature then completely recovered after1d when the seedlings were exposed to thestandard growth conditions. At the same time, the overexpression of CaTIP1-1gene intobacco resulted in higher intercellular CO2concentrations and lower photosynthetic rate during the recovery stage. The overexpression of CaTIP1-1pepper gene in tobacco enhancedthe antioxidant enzyme activities and increased the transcript levels of ROSscavenging-related genes expression under osmotic stresses. Moreover, the viability oftransgenic tobacco cells was higher than in the wild-type after exposure to stress.Overexpression CaTIP1-1gene under osmotic stresses could be beneficial for tobacco stresstolerance.5. CaPIP1-1is located in the plasma intrinsic protein or other subcellular organelles.Three introns, exhibited CT/AC splice junctions, were observed in CaPIP1-1. The numberand location of introns in CaPIP1-1were the same as observed in tomato and potato. Thehigh expression level of this gene was obtained only from fruit. Increased transcript ofCaPIP1-1was found in the different stresses, including chilling stress, salt stress, mannitolstress, ABA treatment and Phytophthora capsici infection. The expression of CaPIP1-1wasdownregulated by50μM HgCl2and100μM fluridone. The pepper plants with silencedCaPIP1-1in Qiemen showed growth inhibition and decreased tolerance to salt and mannitolstresses using detached leaf method.6. Seeds, cultured in modified germination medium (GM3), was more effective than inother media for seedling culture. The shoot was easily induced when the B12explants growon the DM3and DM8medium. While, DM2medium stimulated the bud differentiation fromthe explants of HW203B, on the other hand DM2and DM3, used to enhance budregeneration from cultivar of CK7. The higher number of shoot elongation was observedwhen the buds were moved to the elongation medium after the adventitious shootspre-cultured about14to21d. |
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