1、 3 ) ( y F o y null张null 杰1,2, 周启明2nullnull, 魏江春2*( 1.吉林农业大学中药材学院,长春130118; 2.中国科学院微生物研究所真菌地衣系统学重点实验室,北京100101)K null 1 : 为了从耐旱地衣漠黄梅的共生菌藻基因组中筛选功能基因,并为蛋白质类药物的基因筛选提供平台,采用改进的CTAB方法提取其总DNA,用Sau3Anull限制性内切酶部分酶切基因组DNA,以质粒pUC19为载体, 转入大肠杆菌DH5null中,构建了漠黄梅共生菌藻的宏基因组文库b该文库包含了4null8 105个重组子,插入片段的平均大小为4 kb,覆盖漠黄梅菌
2、藻的整个基因组4次b1 o M : 地衣; pUC19; 大肠杆菌DH5null m s | : Q949null34null null null D S M : A null null null c I | : 1672null3538( 2010) 03null0176null05Construction of Metagenomic Library from Mycobiont and Phycobiontof Desert Lichen Xanthoparmelia deserticolaZHANGJie1, 2, ZHOU Qinullming2* , WEI Jiangnullch
3、un2*(1. College of Chinese Medicinal Materials, JilinAgricultural University, Changchun 130118, China;2. CASKey Laboratory of SystematicMycology andLichenology, Beijing 100101, China)Abstract: In order to obtain the function gene from the Mycobiont and Phycobiont of Xanthoparmeliadenullserticola, wh
4、ich is one of the desert lichens, the metagenomic library has been constructed. In the study,DNA was isolated using improved CTAB method, digested by Sau3A null, integrated into pUC19 vectorand then transformed into E. Coli strain DH5nullto construct metagenomic library, which contains 4null8 105 re
5、combinants. The average length of insertion fragments is 4 kp, the size of the metagenomic covers 4folds of the genome from the mycobiont and phycobiont.Key words: lichen; pUC19; DH5nullnull null v V , F l + ) i F l y b 1989 M Normington 1 ? C I BiP y , ? 4 % 0 ? 2 , 1 F l y B b ) ) l H q / ? 4 7 0
6、l 3null5b2005 M Rosa 6 ,v 7 ) ) I Y y , y V r ? A 4 v 7 F l b“ N , TPS TPP7null8 y b I ) ) 1 B , 3 / , 12 , _ M r T b TaKaRa CIAP 100 nullL 8 “ F “ ,177null 8 null 3 f null : 3 ) ( y F o y37# 30 min “ 5% , 65# 10 min P b M l 8DNAb- 20# i ! b1null2null4null 连接及 CaCl2 法转化大肠杆菌构建基因组文库null CaCl2 E ! v )
7、s % T v D 13 b| ! z s % - 80# i ! b 1 , V 1 Q 8 “ F “ , 16# Q V b65# 10 min P T4 b| 100 nullL s % , |50 nullL ) A p c LB , 37# ! V b ) ,9 8 “ q , K D 1 b| : DNA 8 K D 1 v , s % bV 1null 1 VTable 1. Themix rates of theDNA fragments andthevecnulltor nullL 8 DNA and vector 8 “ I |Ligase system1 2 3DNA
8、DNA fragment 2 4 6 8 pUC19Vector pUC19 2 2 2T4 T4 ligase 1 1 110 T4DNALigase Buffer 1 1 1dH2O 4 2 01null2null5null 文库的扩增null ! 15 cm c LB ! b| v ) p c LB ! , p 200 nullL ) A , 37# ! V b 2 mL c 25% LB ! | ) , | l “ 50 mL 5 , 9 o b- 80# i b1null2null6null 阳性克隆的鉴定及插入片段平均大小的计算null v ) p c LB ! V , G | 1
9、00 ) pUC19 Y M13F M13R Y V PCR bPCR Q : 95# M 5 min; 94# M 1 min, 52# | 30 s, 72# % 4 min, 30Q ;72# % 10 minb _ v l b T L= 1null DNA ( 2 000 6 000 bp)m 1null S au3Anull K = M M 3 ) ( y F mFig. 1. The length of the Mycobiont and PhycobiontDNAfragments digested by Sau3Anull2null3null 8 ! 8 pUC19 BamH
10、nullK =M M , 0null8% j _ ,A U B H ,v l 2 500 3 000 bp, pUC19 v l ( 2 686 bp) M , V DNA $M L DNAb CIAP “ L DNA 5% v , 8 1 2 | 8 “ 764 ) ; 3| 8 “ 328 ) bk 2| 8 “ ( 8 8 1 21) q K , 8 “ c 1null5 ) , y o 1 p b v 2| 1 | y F DNA 8 , S 32 ,i v ) DH5null b2null5null o 9| v ) p c LB ! , p 160 , ! V 4null8 105
11、 ) b 2 mL c 25% LB ! | ) , | l“ 8 50 mL 5 b T N= ln(1- p)/ln(1- f)( ,N o c F X H ; p y F q ;f F X H ( v l y FDNA v l 1 ) 9 o c F X H b o ( v l 4 kbb ) y F v l 1 107 bp,( y F v l 1 108 bp, o , ( y Fv l 9 b9 o c 1null2 105 X H , y F DNA(p= 99%)b o X H 4null8 105, y F 4Q b2null6null X H k pUC19c F y ,
12、# XH ? c LB ! 3 , ) ( V j X H b I n 8 1 , 1 B S PCR _ b ) APCR 0null8% _ , T n m 2bm 2 V A , v l 2 6 kbb G| 100 ) 2 ) PCR 300 bp, b 8 1 , Y V 9 F q 98% , ( v l 4 kbb1 10null PCR PCR products; Mnull Marker(500 15 000 bp)m 2null ) A PCR _ mFig.2. Positive clones detected by PCR3null ) null I ) ( y F o
13、 y 0l I B I ) ( y F o , V 0 l y 4 1 b H ,9 V B + y 3 0 y 4 y % ! b I B v 3 +y 3 , 0 N 14null16 , I 3 7 4 7 ? b V I 3 B , V V I ) ( y F o 0 y , V 7 Y V y ? = 3 , f l , = . S 0 I J . 0 , 1995,26( 5) : 273.16 null , f n . = 6 I J . = v :1 S , 1996, 27( 3) : 384null391.( 175: ) I D : 1 null CABI. Pleute
14、us. http: / / www. Indexfungorum. org/ Names/ Names.asp,2010null01null15. 2 null Kirk P M, Cannon P F, Minter D W, et al. Dictionary of the Funnullgi M . 10 th ed. Wallingford: CAB International, 2008. 3 null | . S ) M . : S , 1963. 4 null z Y . S ) 9 M . : S , 1979. 5 null , , , . ) m M . = : S / , 1986. 6 null 8 , S , . . S 7 3 v ) m k M . : S . K 1 u v ) J . S / , 2008 ( 5): 33null35.30 null 8 null ; c B J . ) , 1988, 7( 2) :89null92.31 null k , z , 6 Y . a1 v ) J . a1j S / , 1997 ( 5) : 1null6.180 ) null null null 2010 M
