GPAT C18 12ACP, PG sn , ;, GPAT C18 12ACP C16 02ACP, 16 0, PG sn21, 4, 9 11, 2000 N ishida 12 GPAT C18 12ACP, GPAT GPAT,,, GPAT,, R

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1 2005, 32 (5) : Acta Horticulturae Sinica (, ) : RT2PCR (Capsicum annuum ) cdna, bp, bp, 463, CaGPA T ;, Northern, CaGPA T,, 8 3 h, 12 h ; 15 CaGPA T : ; ; ; : S : A : X (2005) C lon ing and Expression Ana lysis of Glycerol232phospha te Acyltran sfera se Gene from Sweet Pepper Chen Na, Guo Shangjing, Yan Kun, Dong Xinchun, and Meng Q ingwei 3 ( College of L ife Sciences, Shandong A gricultural U niversity, Taiπan , China) Abstract: A full length cdna of glycerol232phosphate acyltransferase ( GPAT) gene was cloned from sweet pepper by using RT2PCR app roach w ith degenerate p rim ers based on the conserved motifs found in a number of p lant GPAT genes. The CaGPA T is bp encoding 463 am ino acids, with high homology to to2 mato GPAT gene. Phylogenetic tree analysis showed that the am ino acid sequence of GPAT in sweet pepper first gathered with that in tomato, then with that in sp inach. Northern blot showed that the exp ression of CaG2 PA T in leaves was higher than that in other organs such as roots, stem s, petals and fruits. CaGPA T exp ression could be significantly induced by low temperature ( 8 ) for 3 h. But it could not be obviously induced by high temperature ( 35 ). The gene exp ression level had no obvious difference at 5, 10 and 15. These results demonstrate that CaGPA T exp ression is closely correlated w ith p lant responding to low tempera2 ture. Key words: Sweet pepper; Gene clone; Gene exp ression; Low temperature ( PG) 1, PG sn21, 2, 3 PG sn22 4, sn21 3 PG, 5 6 7, ( GPAT) PG, 3 - C Sn ( ), : ; : : ( ) 3 Author for correspondence

2 GPAT C18 12ACP, PG sn , ;, GPAT C18 12ACP C16 02ACP, 16 0, PG sn21, 4, 9 11, 2000 N ishida 12 GPAT C18 12ACP, GPAT GPAT,,, GPAT,, RNA cd NA 25, 30 d, 8 RNA,, g, Trizol RNA ), - 80 ( Trizol RNA, O ligo ( dt), cdna 1, - 80 GPAT GP (5πCAYCARASTgAAgCW g ATCC 3π) GP (5πggAggVggCATDATgTCATR 3π) cdna,, 25 L, 115, 94 4 m in, PCR s, s, s, 35, m in GP (5πgCATATgAATgATgACCCCgAACTTg 3π) GP ( 5πgCCTCCTCATgTTgTCTgTCg CagA 3π), B26 UAP AAP 3π 5π, GP (5πCTCTA AAACCgTCTgCTCCTCTg 3π) GP ( 5πATAACTgCCAAACAAACATCTgC 3π) PCR, PMD182T, DH5, IPTG X2gal, PCR, PCR SalI XbalI, DNAman DNAclub NCB I 112 GPAT RNA, 5π 700 bp Northern , h d, Trizol RNA, 25, 5π 700 bp Northern ( ), 3 h, Northern RT2PCR, cdna 3π cdna 5π cdna cdna, GPA T cdna bp, 5π 51 bp, TAG, 348 ; bp, 463 CaGPA T, GenBank (AY318749)

5 : - 3-825 DNAman, GPAT DNA GPAT DNA, (L ycopersicon escu lentum ) GPAT 8713%, ( Cucum is sa tivus) (Ca rtham us tinctorius) ( Phaseolus vulgaris) (A rabidopsis thaliana) ( Spinacia ol2 eracea) (C

3 5 : DNAman, GPAT DNA GPAT DNA, (L ycopersicon escu lentum ) GPAT 8713%, ( Cucum is sa tivus) (Ca rtham us tinctorius) ( Phaseolus vulgaris) (A rabidopsis thaliana) ( Spinacia ol2 eracea) (C itrus unshiu) GPAT 60%, (Cucu rbita m oscha ta) (O ryza sativa), 5912% 56% 212 C ag PA T cdna 463, GPAT ( 1), GPAT, 8515%, 6017%, ( E laeis guineensis) 5813% 5811% 5712% 5117% 5212%,, GPAT, 1 GPAT : (AY318749) ; : (AY360170) ; : (M80571) ; : (NM197897) ; : (X77370) ; : (Q42713) ; : (BAB79529) ; : (AAF64066) F ig. 1 A lignm en t of pred icted am ino ac id sequences of GPAT genes from d ifferen t plan ts : Capsicum annuum (AY318749) ; : Lycopersicon esculentum (AY360170) ; : Cucum is sativus (M80571) ; : O ryza sativa (NM197897) ; : Spinacia oleracea (X77370) ; : Cartham us tinctorius (Q42713) ; : Citrus unshiu (BAB79529) ; : E laeis guineensis (AAF64066).

826 32, GPAT, GPAT, 1, GPAT N 100, GPAT 213 CaGPAT DNAman GenBank GPAT, CaGPA T,,,, ( 2) 2 GPAT F ig.

3 Expression of CaGPA T in d ifferen t organ s of sweet pepper 1: Leaves; 2: Stem s; 3: Roots; 4: Petals; 5: Fruits.

4 (, 8 ) (, 35 ) CaGPA T 4 Expression of CaGPA T in sweet pepper leaves a t low and h igh tem pera tures for d ifferen t tim e CK: at

4 826 32, GPAT, GPAT, 1, GPAT N 100, GPAT 213 CaGPAT DNAman GenBank GPAT, CaGPA T,,,, ( 2) 2 GPAT F ig. 2 Phylogenetic tree of the deduced am ino ac id sequence of GPAT in d ifferent plants 3 CaGPA T 1: ; 2: ; 3: ; 4: ; 5: F ig. 3 Expression of CaGPA T in d ifferen t organ s of sweet pepper 1: Leaves; 2: Stem s; 3: Roots; 4: Petals; 5: Fruits. 214 C ag PA T CaGPA T 5π cdna Northern, CaGPA T,, ( 3) 25, 8 35, GPAT, 3 h ( 4, ), 35 GPAT ( 4, ) F ig. 4 (, 8 ) (, 35 ) CaGPA T 4 Expression of CaGPA T in sweet pepper leaves a t low and h igh tem pera tures for d ifferen t tim e CK: at 25 ; Left: treatment at 8 ; R ight: treatment at 35 3 h, 25 Northern ( 5),, CaGPA T, RT2PCR F ig. 5 CaGPA T 5 Expression of CaGPA T in sweet pepper leaves trea ted a t d ifferen t tem pera tures for 3 h

5 5 : , GPAT, GPAT, (CaGPA T), GPAT,, GPAT CaGPA T,,,,, CaGPA T, 3 h, 12 h, CaGPA T, - 3 -, CaGPA T,, : 1 N ishida I, Murata N. Chilling sensitivity in p lants and cyanobacteria: The crucial contribution of membrane lip ids. Annu. Rev. Plant Physi2 ol. Plant Mol. B iol., 1996, 47: Murata N, Sato N, Takahashi N, Hamazaki Y. Composition and positional distributions of fatty acids in phospholip ids from leaves of chilling2 sensitive and chilling2resistant p lants. Plant Cell Physiol., 1986, 23: Murata N. Molecular species composition of phosphatidyl2glycerols from chilling2sensitive and chilling2 resistant p lants. Plant Cell Physiol., 1983, 24: Bertram sm, Heinz E. Positional specificity and fatty acid selectivity of purified sn2glycerol 32phosphate acyltransferase from chlorop lasts. Plant Physiol., 1981, 68: Murata N, Isizaki2N ishizawwa O, H igashi S, Hayashi H, Tasaka Y, N ishida I. Genetically engineered alteration in the chilling sensitivity of p lants. Nature, 1992, 356: Wolter F P, Schm idt R, Heinz E. Chilling sensitivity of A robidopsis thaliana with genetically engineered membrane lip ids. EMBO J., 1992, 11: Yokio S, H igashi S I, Kishitani S, Murata N, Toriyama K. Introduction of the cdna for A rabidopsis glycerol232phosphate acyltransferase ( GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice. Molecular B reeding, 1998, 4: A riizum i T, Kishitani S, Inatsugi R, N ishida I, Murata N, Toriyama K. An increase in unsaturation of fatty acids in phosphatidylglycerol from leaves improves the rates of photosynthesis and growth at low temperatures in transgenic rice seedlings. Plant Cell Physiol., 2002, 43 ( 7) : Frentzen M, Heinz E, Mckeon T A, Stumpf P K. Specificities and selectivities of glycerol232phosphate acyltransferase and monoacylglycerol2 32phosphate acyltransferase from pea and sp inach chlorop lasts. Eur. J. B iochem., 1983, 129: Frentzen M, N ishida I, Murata N. Properties of the p lastidial acyl2 ( acyl2carrier2p rotein) : glycerol232phosphate acyltransferase from the chilling sensitive p lant squash (Cucurbita m oschata). Plant Cell Physiol., 1987, 28: W eber S, Wolter F P, Buck F, Frentzen M, Heinz E. Purification and cdna sequencing of an oleate2selective acyl2acp: sn2 glycerol232 phosphate acyltransferase from pea chlorop lasts. PlantMol. B iol., 1991, 17: N ishida I, Sugiura M, Enju A, Nakamura M. A Second gene for acyl2 ( acyl2carrier2p rotein ) : glycerol232phosphate acyltransferase in squash, Cucurbita m oschata cv. Shirogikuza, codes for an oleate2selective isozyme: molecular cloning and p rotein purification studies. Plant and Cell Physiol., 2000, 41 (12) :

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Untitiled 中国农学通报 2009,25(11):117-122 Chinese gricultural Science Bulletin 钙处理对枇杷果实采后生理特性的研究郑国华 ( 福建农林大学园艺学院, 福州 350002) 摘要 : 适当的钙处理可明显减缓枇杷果实的采后生理活力试验结果