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1 36 6 Vol.36, No JOURNAL OF FISHERIES OF CHINA June, 2012 文章编号 : (2012) DOI: /SP.J 坛紫菜色素突变体色素基因的表达定量分析,,,, * (, ) : 6, PCR 4 (Cpeα Cpcα Apcβ Chl.a) ( ), Apcβ>Cpcα>Chl.a>Cpeα, Cpeα,, Cpcα Apcβ Chl.a ;,,, : ; ; ; PCR : Q 785; S 917 : A a(chlorophyll a, Chl.a) (Phycoerythrin, PE) (Phycocyanin, PC) (Allophycocyanin, APC) [1], Chl.a PE PC 3, [2], (Porphyra yezoensis) [3] Miura [4-5], Migita [6] Niwa [7],, [8-9] N- -N'- -N- (NG), 3 [10], 60 Co-γ (P. haitanensis) [11-12] [13] 7, [14] [13-14] Li [15] NG,, Miura [16] Miura [17] Mitman [18], Yan [19], Xie [20],,, ;,, 收稿日期 : 修回日期 : 资助项目 : ( ); ( ) ( ); ( , ); (2010J06016); (JA10186) 通讯作者 :, cschen@jmu.edu.cn

2 6, : 885,,, [21] [22-23], α β, [24] α β,, 18S rrna, PCR (qpcr) ( ), ( ),, : (21±1), 50~60 µmol/(m 2 s), 12 h.. 12 h, 3 3.5~4.5 cm,,,, ( 1) [ (5.0±0.5) cm] ( ) ( 嚢 ) 1.2 坛紫菜总 RNA 分离 0.1 g,, EASYspin RNA (, ) RNA, 1.5% RNA 逆转录 0.5 ml PCR : 2 μg RNA, 20 μmol/l Random Primer 2 μl, RNA 11 μl, 70 2 min, 2 min PCR : 5 RT buffer 4 μl, 10 mmol/l dntp 4 μl, 200 U/μL (PrimeScript R Rtase, TaKaRa)1 μl, 25 5 min, h cdna 20 引物设计 GenBank (DQ449070) (DQ449071) (AY372218) 18S rrna (AB015795) a (AP006715) PCR ( 2) 引物验证 PCR, 10 PCR Buffer 2 μl, 10 mmol/l dntp 1.6 μl, 5 U/μL Taqase 0.1 μl, ddh 2 O 13.3 μl, cdna 1 μl 94 5 min; 94 1 min, s, 72 1 min, 35 ; min, BLAST qpcr 反应条件 ABI-7300 PCR SYBR GreenⅠ qpcr, germplasm no. Tab. 1 表 1 色素突变体不同生长期藻体的选择标准 Selected standard of different growth phases of each pigment mutant pigment mutant ( )/d early phase ( )/d bloom phase ( )/d last phase YSXIII-1 wild 1~3 11~13 21~23 Z-92 breen 1~3 16~18 28~30 Z-103 emerald 1~3 11~13 21~23 Z-71 red 1~3 16~18 26~28 Z-51 red-brown 1~3 11~13 21~23 Z-82 nacarat 1~3 11~13 21~23 YBⅢ purple 1~3 11~13 21~23

3 Tab. 2 表 2 实时荧光定量 PCR 反应引物 Primer sequences for quantitative real-time PCR 引物 primer 引物序列 (5-3 )primer sequences 预期扩增片段长度 /bp predicted size 藻红蛋白 Cpeα 藻蓝蛋白 Cpcα 别藻蓝蛋白 Apcβ 叶绿素 a Chl.a 18S rrna F: TTTGCGTTTGCTCGTGATAGA R: AGCATAGGGCGTTGATAAGGTAA F: AACTCAAATGCCTGGTCCAACT R: CCGTTGCACCTACCACTAAACA F: CGTTATGCTGCATGTATTCGAGA R: TGCACCAATAGGAACACCTAGAGA F: GTTTGGGGTAGTGTATCTCCGTCT R: AAGGAAATCTCTCAGCCATCCA F: TGGAGGGCAAGTCTGGTGA R: CCGACAATTTAGAAGTGTATGATGG , qpcr :20 μl : SYBR R Green Real time PCR Master Mix(ToYoBo)10 μl cdna 2 μl 10 μmol/l 0.8 μl, ddh 2 O 7.2 μl PCR, 95 1 min; s, 57 1 min, s, 60 1 min, s, s,, 3, 标准曲线的绘制 qpcr, CT cdna, , 10 1, , 4 2,, qpcr 坛紫菜色素突变体色素基因表达的 qpcr 分析 qpcr, 3, 4 Pfaffl, ±, (1) [25] ;, (2) C t, target (calibrator test) Etarget Ratio = (1) C t, ref (calibrator test) Eref, C t, target(calibrator test) C t ; C t, ref(calibrator test) C t, E target E ref Ratio = Ct (target, calibrator) E(target, calibrator) (2) E(target, test) Ct(target, test) Ct, ref(calibrator test) Eref, C t(target, calibrator) C t ; C t(target, test) C t ; C t, ref(calibrator test) C t, E (target, calibrator) E (target, test) E ref Excel SPSS13.0, ( ) (LSD), P<0.05, P<0.01 Excel cdna, PCR, qpcr PCR 1,, GenBank, 99%, 96% qpcr 2.2 qpcr, qpcr S,,,,

Cpeα; 2.Cpcα; 3.Apcβ; 4. Chl.a; 5.18S rrna, Cpeα Cpcα Apcβ Chl.a 18S rrna qpcr 83.2 81.9 79.

4 6, : 887 图 1 qpcr 引物 PCR 扩增产物电泳图 Fig. 1 Agarose electrophoresis of PCR amplification with qpcr primers M. DL2000 DNA Marker; 1. Cpeα; 2.Cpcα; 3.Apcβ; 4. Chl.a; 5.18S rrna, Cpeα Cpcα Apcβ Chl.a 18S rrna qpcr ,,, 3,, qpcr 3, 3 qpcr R , K, K E=2 1/k 1, ( 3) 0.94~1.18, 图 2 各色素基因 qpcr 扩增相对标准曲线 Fig. 2 Relative standard curves of qpcr of each gene

5 图 3 坛紫菜色素基因在不同样品间表达水平差异分析 (P<0.05), (P>0.05), (P<0.05), (P>0.05) Fig. 3 The relative expressed levels of each pigment gene in different samples of P. haitanensis Between the different growth phases in the same pigment mutant, bars with different small letters mean significant differences (P<0.05), and the same small letters mean no significant differences (P>0.05); Between the each pigment mutant, bars with different capital letters mean significant differences (P<0.05), and the same capital letters mean no significant differences (P>0.05).

6 6, : 889 表 3 各引物扩增效率 Tab. 3 The amplification efficiency of each primer primers liner regression equation amplification efficiency Cpeα y = x Cpcα y = x Apcβ y = x Chl.a y = x S rrna y = x qpcr 同一色素基因在坛紫菜不同样品间表达水平的差异分析 18S rrna, ( 1,, ( 3) : (1), Cpeα,, (2),, 4 (P>0.05), ( 3) 坛紫菜同一样品内不同色素基因表达水平差异分析 18S rrna, ( Cpeα 1) ( 4),, Apcβ Cpcα, Apcβ> Cpcα>Chl.a>Cpeα 表 4 坛紫菜同一色素突变体内各色素基因表达水平差异分析 Tab. 4 The relative expressed levels of each gene in the same pigment mutant of P. haitanensis pigment mutant gene early phase bloom phase last phase wild Cpcα ± ± ± Apcβ ± ± ± Chl.a 35.12± ± ±9.21 breen Cpcα ± ± ± Apcβ ± ± ± Chl.a 19.11± ± ±19.25 emerald Cpcα ± ± ± Apcβ ± ± ± Chl.a ± ± ±31.22 red Cpcα ± ± ± Apcβ ± ± ± Chl.a ± ± ±10.03 red-brown Cpcα ± ± ± Apcβ ± ± ± Chl.a 31.93± ± ±2.63 nacarat Cpcα ± ± ± Apcβ ± ± ± Chl.a 22.98± ± ±8.57 purple Cpcα ± ± ± Apcβ ± ± ± Chl.a 49.80± ± ±1.92

7 qpcr, qpcr, PCR,,,, [25],,,, [26], : 2 C T, Pfaffl [27] 2 C T, 1 [28] qpcr, RNA, 1,,, PCR, [28], (7, 3, 21 ),, 1( 3),, 2 C T Pfaffl,, [25] Pfaffl 3.2 Chl.a PE PC 3 [2] , Apcβ>Cpcα> Chl.a>Cpeα, Apcβ Cpeα, a,, [2, 29] PCR, 99%, [29] [30] 3,, PE>PC>APC, PE,,, ; PC, PE; APC,,,,,,,,,,

8 6, : 891 : [1],. [J]., 1998, (4): [2],. [J]., 2007, 18(2): [3]. [M]. :, 1994: 72. [4] Miura A. Color variants and heredity of color in Porphyra[J]. Iden (Heredity), 1978, 32(8): [5] Miura A. Genetic analysis of variant color types of light red, light green and light yellow phenotypes of Porphyra yezoensis (Rhodophyta, Bangiaceae) [M].// Hara H, (ed). Origin and evolution of diversity in plant and plant communities. Tokyo: Academia Scientific Book Inc., 1985, [6] Migita S, Fugita Y. Studies on the color mutant types of Porphyra yezoensis Ueda, and their experimental culture[j]. Bulletin of the Faculty of Fisheries,1983, 54: [7] Niwa K, Miura A, Shin J, et al. Characterization and genetic analysis of the violet type pigmentation mutant of Porphyra yezoensis Ueda (Bangiales, Rhodophyta)[J]. Jounal of Korean Society Phycology, 1993, 8(2): [8],,,. I. NG [J]., 2002, 21(5): [9],,,. II. NG [J]., 2003, 22(1): [10],,. [J]., 2000, 24(5): [11],,,. [J]., 2005, 29(2): [12],,. 60 Co-γ (Porphyra yezoensis) [J]., 2009, 40(1): [13],,,. [J]., 2007, 14(3): [14],,,. [J]., 2008, 29(4): [15] Li Y X, Wang G C, Xu P, et al. Induction and characterization of green pigmentation mutant in Porphyra yezoensis Ueda[J]. Aquaculture, 2008, 282(1-4): [16] Miura A, Kunifuji Y. Genetic analysis of the pigmentation types in the seaweed Susabi-nori Porphyra yezoensis[j]. Iden (Heredity), 1980, 34(9) : [17] Miura A, Ohme T M. Mendelian inheritance of pigmentation mutant types in Porphyra yezoensis (Bangiaceae, Rhodophyta) [J]. Janpanese Journal of phycology, 1994, 42: 83. [18] Mitman G G, van der Meer J P. Meiosis, blade development, and sex determination in Porphyra purpurea (Rhodophyta) [J]. Journal of Phycology, 1994, 30(1): [19] Yan X H, Li L, Aruga Y. Genetic analysis of the position of meiosis in Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta) [J]. Journal of Applied Phycology, 2005, 17(6): [20] Xie C T, Chen C S, Xu Y, et al. Construction of a genetic linkage map for Porphyra haitanensis (Bangiales, Rhodophyta) Based on sequence-related amplified polymorphism and simple sequence repeat markers[j]. Journal of Phycology, 2010, 46(4): [21],,,. α, β [J]., 2005, 27(2): [22],,,. α, β [J]., 2007, 29(2): [23]. [D]., [24],,,. α β [J]., 2011, 27(1): [25] Whelan J A, Russell N B, Whelan M A A method for the absolute quantification of cdna using real-time PCR[J]. Journal of Immunological Methods, 2003, 278(1-2): [26] Nolan T, Hands R E, Bustin S A. Quantification of mrna using real-time RT-PCR[J]. Nature Protocols, 2006, 1(3): [27] Yuan J S, Reed A, Chen F, et al. Statistical analysis of real-time PCR data[j] BMC Bioinformatics, 2006, 7: 85. [28] Yuan J S, Wang D, Stewart C, et al. Statistical methods for efficiency adjusted real-time PCR quantification[j]. Biotechnol Journal, 2008, 3(1): [29],,,. [J]., 2006, 42(1): 1 9. [30]. [J]., 1993, 24(6):

9 Quantitative analysis of pigment gene expression in pigment mutants of Porphyra haitanensis HUANG Hui-zhen, XIE Chao-tian, JI De-hua, XU Yan, CHEN Chang-sheng * (College of Fisheries, Jimei University, Xiamen , China) Abstract: Phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), and chlorophyll a(chl.a) are the four major photosynthetic pigments of Porphyra haitanensis. Their contents and ratio determine the blade color of Porphyra. In this study, the relative expressed levels of four pigment genes (Cpeα, Cpcα, Apcβ, and Chl.a) in different growth phases (early phase, bloom phase, and final phase) were determined using quantitative real-time PCR (qpcr) in one wild line and six pigment mutant lines of P. haitanensis. In all the lines, the relative expression levels of the four pigment genes were in the sequence: Apcβ>Cpcα> Chl.a>Cpeα. The expressed level of Cpeα varied significantly in the different phases in all the lines; however, the expressions of Cpcα, Apcβ, and Chl.a were stable in all phases in all lines. Furthermore, liner correlation and regression analysis indicated that the expression levels of the pigment genes had no relation to the pigment protein contents in any of the lines. In other words, the expression levels of the four pigment genes had no relation to the differences in blade color among the Porphyra lines. Based on these results, we speculate that the pigment mutation of P. haitanensis was induced by mutations in genes that affect the synthesis of the pigment proteins. Key words: Porphyra haitanensis; pigment mutant; pigment gene; quantitative real-time PCR Corresponding author: CHEN Chang-sheng. cschen@jmu.edu.cn

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吉林农业大学学报２０１６３８１８７９１ＪｏｕｒｎａｌｏｆＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｘｕｅｂａｏｊｌａｕｅｄｕｃｎＥｍａｉｌｊｌｎｄｘｂｖｉｐｓｉｎａｃｏｍ神经生长因子ｍｒｎａ在鹅等级前卵泡中的表达刘璐璐隋玉健朱海洋武惠岩杨辉孙永峰吉林农业大学动物科学技术学院长春１３０１１８