CN110499319A - 一种通过基因编辑技术提高番茄中番茄红素含量的方法 - Google Patents

一种通过基因编辑技术提高番茄中番茄红素含量的方法 Download PDF

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CN110499319A
CN110499319A CN201910899703.5A CN201910899703A CN110499319A CN 110499319 A CN110499319 A CN 110499319A CN 201910899703 A CN201910899703 A CN 201910899703A CN 110499319 A CN110499319 A CN 110499319A
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汪启明
周池
饶力群
陈苗
朱建华
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Abstract

本发明公开了一种基因编辑技术提高番茄中番茄红素含量的方法，包括以下步骤：分别构建Lcyb1、Lcyb2、CycB和LcyE基因的靶序列T1～T4，然后通过靶序列分别构建其对应的T1sgRNA～T4sgRNA表达盒；将T1sgRNA～T4sgRNA表达盒与crispr/cas9表达载体进行组装，得到重组质粒；利用农杆菌侵染法将重组质粒转化到番茄中，得到番茄新材料，因为新番茄材料中与番茄红素环化酶合成相关基因会存在一个或多个基因的突变，从而抑制番茄红素环化酶的合成，使得新番茄材料的番茄红素含量提高；本发明的方法可实现对一个或多个番茄红素环化酶基因进行编辑，且通过遗传分离可筛选出不含外源载体序列的番茄材料，不存在转基因风险。

Description

一种通过基因编辑技术提高番茄中番茄红素含量的方法
技术领域
本发明属于基因工程技术领域，具体涉及一种通过基因编辑技术提高番茄中番茄红素含量的方法。
背景技术
番茄红素是成熟番茄的主要色素，是一种不含氧的类胡萝卜素，也是目前在自然界的植物中被发现的最强抗氧化剂之一。科学证明，人体内的单线态氧和氧自由基是侵害人体自身免疫系统的罪魁祸首，番茄红素清除自由基的功效远胜于其他类胡萝卜素和维生素E，其淬灭单线态氧速率常数是维生素E的100倍。由于其优越的生理功能，它不仅可以有效的防治因衰老，免疫力下降引起的各种疾病，还具有预防心血管疾病、动脉硬化等作用，进一步而言，其对抗癌抑癌也有一定的功效，因此，它受到世界各国专家的关注。
番茄红素最早是在番茄中发现，因而取名番茄红素，提高番茄中番茄红素的产量无论是番茄营养组份的提高还是用于番茄红素的提取都有很大作用。
CRISPR(Clustered regularly interspaced short palindromic repeats)是原核生物基因组内的一段重复序列，是生命进化历史上，细菌和病毒进行斗争产生的免疫武器。科学家掌握了细菌拥有多种切除外来病毒基因的免疫功能，其中比较典型的模式是依靠一个复合物，该复合物能在一段RNA指导下，定向寻找目标DNA序列，然后将该序列进行切除。许多细菌免疫复合物都相对复杂，其中科学家掌握了对一种蛋白Cas9的操作技术，并先后对多种目标细胞DNA进行切除，这就是crispr/cas9基因编辑系统，是继“锌指核酸内切酶(ZFN)”、“类转录激活因子效应物核酸酶(TALEN)”之后出现的第三代基因组定点编辑技术。
番茄中番茄红素的含量主要跟番茄红素环化酶有关，而番茄环化酶的合成跟4条染色体上的4个基因相关，分别为Lcyb1(4号染色体)、Lcyb2(10号染色体)、CycB(6号染色体)和LcyE(12号染色体)。但是目前并未有通过调整上述基因提高番茄中番茄红素的研究。
发明内容
本发明的目的是提供一种通过crispr/cas9基因编辑技术提高番茄中番茄红素含量的方法，其可通过改变与番茄红素环化酶合成相关的一个或多个基因，达到抑制番茄红素环化酶的合成，从而提高番茄红素含量的目的。
本发明这种通过crispr/cas9基因编辑技术提高番茄中番茄红素含量的方法，包括以下步骤:
1)分别构建Lcyb1、Lcyb2、CycB和LcyE基因的靶序列T1～T4，然后通过靶序列分别构建其对应的T1sgRNA～T4 sgRNA表达盒；
2)将步骤1)中的T1sgRNA～T4sgRNA表达盒与crispr/cas9表达载体进行组装，得到重组质粒；
3)利用农杆菌侵染法将步骤2)中的重组质粒转化到番茄中，得到番茄新材料，因为新番茄材料中与番茄红素环化酶合成相关基因会存在一个或多个基因的突变，从而抑制番茄红素环化酶的合成，使得新番茄材料的番茄红素含量高；
其中：T1～T4的核苷酸序列如序列1～4所示。
所述步骤1)中，T1sgRNA表达盒中含有T1sgRNA序列如序列5所示；T2sgRNA表达盒中含有T2sgRNA序列如序列6所示；T3sgRNA表达盒中含有T3sgRNA序列如序列7所示；T4sgRNA表达盒中含有T4sgRNA序列如序列8所示；
所述步骤2)中，所述的重组质粒的核苷酸序列如序列9所示。
所述的靶序列T1～T4在制备高番茄红素的番茄材料中的应用。
所述的T1sgRNA～T4sgRNA序列在制备高番茄红素的番茄材料中的应用。
所述的重组载体在制备高番茄红素的番茄材料中的应用。
本发明的有益效果：1)本发明构建了番茄中与番茄红素环化酶相关基因的靶序列、sgRNA和crispr/cas9重组载体，可用于培育番茄红素含量高的番茄新材料。2)本发明的方法可实现对一个或多个番茄红素环化酶相关基因进行编辑，且通过遗传分离可筛选出不含外源载体序列的番茄材料，不存在转基因风险。
附图说明
图1实施例2中的组装路线图；
图2实施例2中的阳性克隆电泳图；
图3实施例3中#10基因序列突变图；
图4实施例3中#119基因序列突变图；
图5实施例3中野生番茄果实与#10番茄果实的对比图。
具体实施方式
实施例1 sgRNA表达盒的构建
通过番茄数据库找到四个基因的序列号：LcyB1(Solyc04g040190.1)、LcyB2(Solyc10g079480.1)、CycB(chromosome 6)、LcyE(Solyc12g008980.1)。
通过使用CRISPR-P(http://cbi.hzau.edu.cn/crispr/)进行靶位点设计，得出：
LcyB1的靶序列T1为：GTACACTGCTGCACCAGACC(序列1)；LcyB2的靶序列T2为：GCACAGCAGGTTTCGGAGGC(序列2)；CycB的靶序列T3为：ATTATCGGAGCTGGCCCTGC(序列3)；LcyE的靶序列T4为GCCGCGGAGTCAGCTAAATT(序列4)。
根据靶序列的首个碱基序列确定使用的启动子，其中T1靶序列的启动子为U6-1，T2靶序列的启动子为U6-29，T1靶序列的启动子U3b，T4靶序列的启动子U6-1；并根据靶序列和启动子设计相应的引物序列。
表1引物序列表
采用常规PCR和Overlapping PCR构建sgRNA表达盒
T1sgRNA表达盒的构建：
第一轮PCR反应体系为：U-F：1ul，OL-C11 Rev：0.5ul，OL-C12 For：0.5ul，gR-R：1ul，pYLsgRNA-AtU6-1载体(其具体序列如序列28所示)：1ul Add PCR mix to 50ul进行第一轮反应，得到第一轮反应产物。
将第一轮反应产物稀释10倍后，取1ul第一轮稀释后的反应产物，Bsa-C1For：0.5ul，Bsa-C1 Rev：0.5ul，然后Add PCR mix to 30ul进行第二轮反应，即得T1sgRNA表达盒，测得其序列如序列5所示，序列5中包含有U6-1启动子，T1靶序列以及保守序列。
T2sgRNA表达盒的构建：
第一轮PCR反应体系为：U-F：1ul，OL-C21 Rev：0.5ul，OL-C22 For：0.5ul，gR-R：1ul，pYLsgRNA-AtU6-29载体(其具体序列如序列29所示)：1ul Add PCR mix to 50ul进行第一轮反应，得到第一轮反应产物。
将第一轮反应产物稀释10倍后，取1ul第一轮稀释后的反应产物，Bsa-C2For：0.5ul，Bsa-C2 Rev：0.5ul，然后Add PCR mix to 30ul进行第二轮反应，即得T2sgRNA表达盒，测得其序列如序列6所示，序列6中包含有U6-29启动子，T2靶序列以及保守序列。
T3sgRNA表达盒的构建：
第一轮PCR反应体系为：U-F：1ul，OL-C31 Rev：0.5ul，OL-C32 For：0.5ul，gR-R：1ul，pYLsgRNA-AtU3b载体(其具体序列如序列30所示)：1ul，Add PCR mix to 50ul进行第一轮反应，得到第一轮反应产物。
将第一轮反应产物稀释10倍后，取1ul第一轮稀释后的反应产物，Bsa-C2For：0.5ul，Bsa-C2 Rev：0.5ul，然后Add PCR mix to 30ul进行第二轮反应，即得T3sgRNA表达盒，测得其序列如序列7所示，序列7中包含有U3b启动子，T3靶序列以及保守序列。
T4sgRNA表达盒的构建：
第一轮PCR反应体系为：U-F：1ul，OL-C41 Rev：0.5ul，OL-C42 For：0.5ul，gR-R：1ul，pYLsgRNA-AtU6-1载体(其具体序列如序列28所示)：1ul Add PCR mix to 50ul进行第一轮反应，得到第一轮反应产物。
将第一轮反应产物稀释10倍后，取1ul第一轮稀释后的反应产物，Bsa-C4For：0.5ul，Bsa-C4 Rev：0.5ul，然后Add PCR mix to 30ul进行第二轮反应，即得T2sgRNA表达盒，测得其序列如序列8所示，序列8中包含有U6-1启动子，T4靶序列以及保守序列。
实施例2组装sgRNA到crispr/cas9表达载体
采用Golden Gate Cloning方法进行组装，其组装路线如图1所示，反应体系如表2所示。
表2
按照表2的反应体系，将pYLCRISPR/Cas9质粒与T1～T4sgRNA表达盒的酶切-连接反应体系进行酶切、连接。其具体反应参数如下：先3cycles(37℃ for10min,10℃ for 5min，20℃ for 5min)；再10cycles(37℃ for 3min,10℃ for5min,20℃ for 5min)，最后37℃for 5min，得到连接产物。
将连接产物进行转化并涂Amp+平板，通过测序引物序列31和序列32鉴定阳性克隆，
Pubi-F1:5’-GCGGTGTCATCTATGTTACTAG(序列31)
SP-R:5’-CCCGACATAGATGCAATAACTTC(序列32)
其结果如图2所示，17组连接产物中(18为maker)，在4号组中检测到有一个约2000bp阳性克隆序列(序列33)，命名为pSL-Pubi-H-4-4，测得pSL-Pubi-H-4-4中含有T1sgRNA～T4 sgRNA的序列(图2所示)。
用质粒抽提试剂盒提取含阳性克隆质粒，即为重组质粒。
实施例3
将实施例2中的重组质粒转化到农杆菌菌株中，然后采用农杆菌介导的侵染番茄愈伤组织，通过农杆菌中的Ti质粒，将重组载体的T-DNA切割下来，插入到番茄基因组中，通过再生获得转基因番茄苗，待转基因苗长大后提取DNA进行阳性转基因植物和有编辑效果转基因株系筛选，筛选阳性转基因植物采用筛选阳性克隆载体引物进行PCR，产物电泳检测，有条带则为阳性转基因植物。
对阳性转基因植物还要进行基因编辑效果的检测，共检测到二个转基因株系具有编辑效果，分别为#10CycB基因：自5’端开始272处一个碱基C缺失；Lcyb2基因自5’端开始第311-313处三个碱基AGG缺失(如图3所示)。#119Lcyb1基因：自5’端开始第434处插入一个碱基A；Lcyb2基因自5’端开始第302-330处二十九个碱基AGGTTTCGGAGGCTGGGTTATCGGTTTGC缺失(如图4所示)。
将上述转基因植株#10检测其番茄果实中番茄红素含量，对比为野生型，取同一发育阶段的果实，进行番茄红素的测定，其结果如图5所示，最终#10的平均番茄红素含量为#10为159.5mg/g、野生型为72.5mg/g。
.逆序列表
<110> 湖南农业大学
<120> 一种通过基因编辑技术提高番茄中番茄红素含量的方法
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atctaaacaa atcttgttgt atatataaca ctgagggagc aacattggtc attatcggag 360
ctggccctgc gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac 420
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<213> 重组质粒(人工序列)
<400> 9
tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 60
gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact 120
ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac 180
atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc 240
cttatctggg aactactcac acattattat ggagaaactc gagcttgtcg atcgacagat 300
cccggtcggc atctactcta tttctttgcc ctcggacgag tgctggggcg tcggtttcca 360
ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt 420
tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc 480
caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg 540
cggagcatat acgcccggag tcgtggcgat cctgcaagct ccggatgcct ccgctcgaag 600
tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc 660
tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca 720
ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg 780
cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg 840
tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca 900
cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg 960
ctaagatcgg ccgcagcgat cgcatccata gcctccgcga ccggttgtag aacagcgggc 1020
agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag 1080
gtcaggctct cgctaaactc cccaatgtca agcacttccg gaatcgggag cgcggccgat 1140
gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc 1200
aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag 1260
agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc 1320
gcggtgagtt caggcttttt catatctcat tgccccccgg gatctgcgaa agctcgagag 1380
agatagattt gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact 1440
tccttatata gaggaaggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca 1500
gtggagatat cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc 1560
acgatgctcc tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga 1620
acgatagcct ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact 1680
gtccttttga tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt 1740
accctttgtt gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc 1800
ttggagtaga cgagagtgtc gtgctccacc atgttatcac atcaatccac ttgctttgaa 1860
gacgtggttg gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg 1920
ggaccactgt cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat 1980
ttgtaggtgc caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa 2040
tggaatccga ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg 2100
tcttctgaga ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg 2160
ttggcaagct gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 2220
aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2280
atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 2340
tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 2400
acgaattcga gctcggtacc aactataacg gtcctaaggt agcgaaggat ccgctcgcta 2460
ccttaagaga ggatatccct ccatcctata atgtaggcta taggaactag ggcaaggccg 2520
gccatgcggc cgcaagctgg gtgcagcgtg acccggtcgt gcccctctct agagataatg 2580
agcattgcat gtctaagtta taaaaaatta ccacatattt tttttgtcac acttgtttga 2640
agtgcagttt atctatcttt atacatatat ttaaacttta ctctacgaat aatataatct 2700
atagtactac aataatatca gtgttttaga gaatcatata aatgaacagt tagacatggt 2760
ctaaaggaca attgagtatt ttgacaacag gactctacag ttttatcttt ttagtgtgca 2820
tgtgttctcc tttttttttg caaatagctt cacctatata atacttcatc cattttatta 2880
gtacatccat ttagggttta gggttaatgg tttttataga ctaatttttt tagtacatct 2940
attttattct attttagcct ctaaattaag aaaactaaaa ctctatttta gtttttttat 3000
ttaataattt agatataaaa tagaataaaa taaagtgact aaaaattaaa caaataccct 3060
ttaagaaatt aaaaaaacta aggaaacatt tttcttgttt cgagtagata atgccagcct 3120
gttaaacgcc gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg 3180
gccaagcgaa gcagacggca cggcatctct gtcgctgcct ctggacccct ctcgagagtt 3240
ccgctccacc gttggacttg ctccgctgtc ggcatccaga aattgcgtgg cggagcggca 3300
gacgtgagcc ggcacggcag gcggcctcct cctcctctca cggcacggca gctacggggg 3360
attcctttcc caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc 3420
cctccacacc ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat 3480
ctcccccaaa tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc 3540
cccccctctc taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct 3600
acttctgttc atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg 3660
tacacggatg cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct 3720
ttggggaatc ctgggatggc tctagccgtt ccgcagacgg gatcgatttc atgatttttt 3780
ttgtttcgtt gcatagggtt tggtttgccc ttttccttta tttcaatata tgccgtgcac 3840
ttgtttgtcg ggtcatcttt tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg 3900
ttgggcggtc gttctagatc ggagtagaat tctgtttcaa actacctggt ggatttatta 3960
attttggatc tgtatgtgtg tgccatacat attcatagtt acgaattgaa gatgatggat 4020
ggaaatatcg atctaggata ggtatacatg ttgatgcggg ttttactgat gcatatacag 4080
agatgctttt tgttcgcttg gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt 4140
tctagatcgg agtagaatac tgtttcaaac tacctggtgt atttattaat tttggaactg 4200
tatgtgtgtg tcatacatct tcatagttac gagtttaaga tggatggaaa tatcgatcta 4260
ggataggtat acatgttgat gtgggtttta ctgatgcata tacatgatgg catatgcagc 4320
atctattcat atgctctaac cttgagtacc tatctattat aataaacaag tatgttttat 4380
aattattttg atcttgatat acttggatga tggcatatgc agcagctata tgtggatttt 4440
tttagccctg ccttcatacg ctatttattt gcttggtact gtttcttttg tcgatgctca 4500
ccctgttgtt tggtgttact tctgcagatg gctcctaaga agaagcggaa ggttggtatt 4560
cacggggtgc ctgcggctga caagaagtac tccatcggcc tcgacatcgg caccaacagc 4620
gtcggctggg cggtgatcac cgacgagtac aaggtcccgt ccaagaagtt caaggtcctg 4680
ggcaacaccg accgccactc catcaagaag aacctcatcg gcgccctcct cttcgactcc 4740
ggcgagacgg cggaggcgac ccgcctcaag cgcaccgccc gccgccgcta cacccgccgc 4800
aagaaccgca tctgctacct ccaggagatc ttctccaacg agatggcgaa ggtcgacgac 4860
tccttcttcc accgcctcga ggagtccttc ctcgtggagg aggacaagaa gcacgagcgc 4920
caccccatct tcggcaacat cgtcgacgag gtcgcctacc acgagaagta ccccactatc 4980
taccaccttc gtaagaagct tgttgactct actgataagg ctgatcttcg tctcatctac 5040
cttgctctcg ctcacatgat caagttccgt ggtcacttcc ttatcgaggg tgaccttaac 5100
cctgataact ccgacgtgga caagctcttc atccagctcg tccagaccta caaccagctc 5160
ttcgaggaga accctatcaa cgcttccggt gtcgacgcta aggcgatcct ttccgctagg 5220
ctctccaagt ccaggcgtct cgagaacctc atcgcccagc tccctggtga gaagaagaac 5280
ggtcttttcg gtaacctcat cgctctctcc ctcggtctga cccctaactt caagtccaac 5340
ttcgacctcg ctgaggacgc taagcttcag ctctccaagg atacctacga cgatgatctc 5400
gacaacctcc tcgctcagat tggagatcag tacgctgatc tcttccttgc tgctaagaac 5460
ctctccgatg ctatcctcct ttcggatatc cttagggtta acactgagat cactaaggct 5520
cctctttctg cttccatgat caagcgctac gacgagcacc accaggacct caccctcctc 5580
aaggctcttg ttcgtcagca gctccccgag aagtacaagg agatcttctt cgaccagtcc 5640
aagaacggct acgccggtta cattgacggt ggagctagcc aggaggagtt ctacaagttc 5700
atcaagccaa tccttgagaa gatggatggt actgaggagc ttctcgttaa gcttaaccgt 5760
gaggacctcc ttaggaagca gaggactttc gataacggct ctatccctca ccagatccac 5820
cttggtgagc ttcacgccat ccttcgtagg caggaggact tctacccttt cctcaaggac 5880
aaccgtgaga agatcgagaa gatccttact ttccgtattc cttactacgt tggtcctctt 5940
gctcgtggta actcccgttt cgcttggatg actaggaagt ccgaggagac tatcacccct 6000
tggaacttcg aggaggttgt tgacaagggt gcttccgccc agtccttcat cgagcgcatg 6060
accaacttcg acaagaacct ccccaacgag aaggtcctcc ccaagcactc cctcctctac 6120
gagtacttca cggtctacaa cgagctcacc aaggtcaagt acgtcaccga gggtatgcgc 6180
aagcctgcct tcctctccgg cgagcagaag aaggctatcg ttgacctcct cttcaagacc 6240
aaccgcaagg tcaccgtcaa gcagctcaag gaggactact tcaagaagat cgagtgcttc 6300
gactccgtcg agatcagcgg cgttgaggac cgtttcaacg cttctctcgg tacctaccac 6360
gatctcctca agatcatcaa ggacaaggac ttcctcgaca acgaggagaa cgaggacatc 6420
ctcgaggaca tcgtcctcac tcttactctc ttcgaggata gggagatgat cgaggagagg 6480
ctcaagactt acgctcatct cttcgatgac aaggttatga agcagctcaa gcgtcgccgt 6540
tacaccggtt ggggtaggct ctcccgcaag ctcatcaacg gtatcaggga taagcagagc 6600
ggcaagacta tcctcgactt cctcaagtct gatggtttcg ctaacaggaa cttcatgcag 6660
ctcatccacg atgactctct taccttcaag gaggatattc agaaggctca ggtgtccggt 6720
cagggcgact ctctccacga gcacattgct aaccttgctg gttcccctgc tatcaagaag 6780
ggcatccttc agactgttaa ggttgtcgat gagcttgtca aggttatggg tcgtcacaag 6840
cctgagaaca tcgtcatcga gatggctcgt gagaaccaga ctacccagaa gggtcagaag 6900
aactcgaggg agcgcatgaa gaggattgag gagggtatca aggagcttgg ttctcagatc 6960
cttaaggagc accctgtcga gaacacccag ctccagaacg agaagctcta cctctactac 7020
ctccagaacg gtagggatat gtacgttgac caggagctcg acatcaacag gctttctgac 7080
tacgacgtcg accacattgt tcctcagtct ttccttaagg atgactccat cgacaacaag 7140
gtcctcacga ggtccgacaa gaacaggggt aagtcggaca acgtcccttc cgaggaggtt 7200
gtcaagaaga tgaagaacta ctggaggcag cttctcaacg ctaagctcat tacccagagg 7260
aagttcgaca acctcacgaa ggctgagagg ggtggccttt ccgagcttga caaggctggt 7320
ttcatcaaga ggcagcttgt tgagacgagg cagattacca agcacgttgc tcagatcctc 7380
gattctagga tgaacaccaa gtacgacgag aacgacaagc tcatccgcga ggtcaaggtg 7440
atcaccctca agtccaagct cgtctccgac ttccgcaagg acttccagtt ctacaaggtc 7500
cgcgagatca acaactacca ccacgctcac gatgcttacc ttaacgctgt cgttggtacc 7560
gctcttatca agaagtaccc taagcttgag tccgagttcg tctacggtga ctacaaggtc 7620
tacgacgttc gtaagatgat cgccaagtcc gagcaggaga tcggcaaggc caccgccaag 7680
tacttcttct actccaacat catgaacttc ttcaagaccg agatcaccct cgccaacggc 7740
gagatccgca agcgccctct tatcgagacg aacggtgaga ctggtgagat cgtttgggac 7800
aagggtcgcg acttcgctac tgttcgcaag gtcctttcta tgcctcaggt taacatcgtc 7860
aagaagaccg aggtccagac cggtggcttc tccaaggagt ctatccttcc aaagagaaac 7920
tcggacaagc tcatcgctag gaagaaggat tgggacccta agaagtacgg tggtttcgac 7980
tcccctactg tcgcctactc cgtcctcgtg gtcgccaagg tggagaaggg taagtcgaag 8040
aagctcaagt ccgtcaagga gctcctcggc atcaccatca tggagcgctc ctccttcgag 8100
aagaacccga tcgacttcct cgaggccaag ggctacaagg aggtcaagaa ggacctcatc 8160
atcaagctcc ccaagtactc tcttttcgag ctcgagaacg gtcgtaagag gatgctggct 8220
tccgctggtg agctccagaa gggtaacgag cttgctcttc cttccaagta cgtgaacttc 8280
ctctacctcg cctcccacta cgagaagctc aagggttccc ctgaggataa cgagcagaag 8340
cagctcttcg tggagcagca caagcactac ctcgacgaga tcatcgagca gatctccgag 8400
ttctccaagc gcgtcatcct cgctgacgct aacctcgaca aggtcctctc cgcctacaac 8460
aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct cttcacgctc 8520
acgaacctcg gcgcccctgc tgctttcaag tacttcgaca ccaccatcga caggaagcgt 8580
tacacgtcca ccaaggaggt tctcgacgct actctcatcc accagtccat caccggtctt 8640
tacgagactc gtatcgacct ttcccagctt ggtggtgata agcgtcctgc tgccaccaaa 8700
aaggccggac aggctaagaa aaagaagtag gatcctcccg atcgttcaaa catttggcaa 8760
taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg 8820
ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgaggtgg 8880
gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag 8940
cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggagcac 9000
cggtaaggcg cgccgtagtg ctcgactagt atggaatcgg cagcaaagga gaaatctcaa 9060
aattccggca gaacaatttt gaatctcgat ccgtagaaac gagacggtca ttgttttagt 9120
tccaccacga ttatatttga aatttacgtg agtgtgagtg agacttgcat aagaaaataa 9180
aatctttagt tgggaaaaaa ttcaataata taaatgggct tgagaaggaa gcgagggata 9240
ggcctttttc taaaataggc ccatttaagc tattaacaat cttcaaaagt accacagcgc 9300
ttaggtaaag aaagcagctg agtttatata tggttagaga cgaagtagtg attgtacact 9360
gctgcaccag accgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 9420
aacttgaaaa agtggcaccg agtcggtgct ttttttcaag agcttggagt ggatggaccc 9480
tgacactgga atcggcagca aaggaaaata tcagagatct cttacagtta gtttcgttct 9540
taatccaaac tactgcagcc tgacagacaa atgaggatgc aaacaatttt aaagtttatc 9600
taacgctagc tgttttgttt cttctctctg gtgcaccaac gacggcgttt tctcaatcat 9660
aaagaggctt gttttactta aggccaataa tgttgatgga tcgaaagaag agggctttta 9720
ataaacgagc ccgtttaagc tgtaaacgat gtcaaaaaca tcccacatcg ttcagttgaa 9780
aatagtagct ctgtttatat attggtagag tcgactaaga gattgcacag caggtttcgg 9840
aggcgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 9900
aagtggcacc gagtcggtgc tttttttcaa gagcttggag tggatggagt gaagactttg 9960
gaatcggcag caaaggattt actttaaatt ttttcttatg cagcctgtga tggataactg 10020
aatcaaacaa atggcgtctg ggtttaagaa gatctgtttt ggctatgttg gacgaaacaa 10080
gtgaactttt aggatcaact tcagtttata tatggagctt atatcgagca ataagataag 10140
tgggcttttt atgtaattta atgggctatc gtccatagat tcactaatac ccatgcccag 10200
tacccatgta tgcgtttcat ataagctcct aatttctccc acatcgctca aatctaaaca 10260
aatcttgttg tatatataac actgagggag caacattggt cattatcgga gctggccctg 10320
cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag 10380
tggcaccgag tcggtgcttt ttttcaagag cttggagtgg atggaaagga ctacatggaa 10440
tcggcagcaa aggagaaatc tcaaaattcc ggcagaacaa ttttgaatct cgatccgtag 10500
aaacgagacg gtcattgttt tagttccacc acgattatat ttgaaattta cgtgagtgtg 10560
agtgagactt gcataagaaa ataaaatctt tagttgggaa aaaattcaat aatataaatg 10620
ggcttgagaa ggaagcgagg gataggcctt tttctaaaat aggcccattt aagctattaa 10680
caatcttcaa aagtaccaca gcgcttaggt aaagaaagca gctgagttta tatatggtta 10740
gagacgaagt agtgattgcc gcggagtcag ctaaattgtt ttagagctag aaatagcaag 10800
ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 10860
caagagcttg gagtggatgg atacgcgtcg gtatcattgg cgcgcctctc gagctagcgg 10920
ccgcatgcat cgatctccta catcgtataa attagcctat acgaagttat tgcatctatg 10980
tcgggtgcgg agaaagaggt aatgaaatgg cagtattaga tctgataact tcgtataatg 11040
tatgctatac gaagttatga ctgcaggtcg acacccataa tagctgtttg ccaagcttgg 11100
cactggccgt cgtttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 11160
cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 11220
cgcccttccc aacagttgcg cagcctgaat ggcgaatgct agagcagctt gagcttggat 11280
cagattgtcg tttcccgcct tcagtttaaa ctatcagtgt ttgacaggat atattggcgg 11340
gtaaacctaa gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag 11400
gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa 11460
gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc 11520
cgtcttctga aaacgacatg tcgcacaagt cctaagttac gcgacaggct gccgccctgc 11580
ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact 11640
agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg ggctatgccc 11700
gcgtcagcac cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct 11760
gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg gagctggcca 11820
ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg 11880
cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc 11940
tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc atggtgttga 12000
ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg 12060
ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg 12120
cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg 12180
ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag 12240
tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg 12300
acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga 12360
cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg 12420
gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg 12480
tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg 12540
ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc 12600
gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct 12660
gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc 12720
ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc 12780
gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg 12840
attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc 12900
caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg 12960
cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc 13020
attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc 13080
acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag 13140
tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt 13200
gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa 13260
tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa 13320
gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca 13380
cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga 13440
tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc 13500
taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc 13560
ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga 13620
acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga 13680
acccccaagc ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg 13740
cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca 13800
acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg 13860
caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg 13920
cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg 13980
cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt 14040
gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc 14100
cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa 14160
ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga 14220
cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac 14280
ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg 14340
ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag 14400
cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat 14460
cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc 14520
cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag 14580
atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg 14640
tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga 14700
ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc 14760
atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa 14820
aggtcgaaaa ggtctgtttc ctgtggatag cacgtacatt gggaacccaa agccgtacat 14880
tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat 14940
gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact 15000
tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga 15060
agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg 15120
tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc 15180
agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct 15240
gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 15300
tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 15360
gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 15420
ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 15480
aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 15540
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 15600
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 15660
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 15720
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 15780
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 15840
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 15900
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 15960
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 16020
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 16080
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 16140
tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 16200
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 16260
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 16320
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta 16380
ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc 16440
cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg 16500
gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa 16560
gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa 16620
gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt 16680
aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa 16740
gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc 16800
gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg 16860
ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct 16920
ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca 16980
tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt 17040
catttttaaa tataggtttt cattttctcc caccagctta tataccttag caggagacat 17100
tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat 17160
tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa 17220
gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa 17280
taccagaaaa cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg 17340
gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa 17400
catgctaccc tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc 17460
cgaatagcat cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc 17520
cgtcccggac tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg 17580
ggagctgttg gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac 17640
aacttaataa cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg 17700
ggatctggat tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt 17760
attttacaaa tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc 17820
ctataggaac cctaattccc ttatctggga actactcaca cattattatg gagaaactcg 17880
agcttgtcga tcgacagatc ccggtcggca tctactctat ttctttgccc tcggacgagt 17940
gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc 18000
cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc 18060
gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata 18120
gagttggtca agaccaatgc ggagcatata cgcccggagt cgtggcgatc ctgcaagctc 18180
cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca 18240
gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat 18300
gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac 18360
gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc 18420
gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc 18480
aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg 18540
gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatag cctccgcgac 18600
cggttgtaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc 18660
acggcgggag atgcaatagg tcaggctctc gctaaactcc ccaatgtcaa gcacttccgg 18720
aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc 18780
ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg 18840
agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag 18900
aaacttctcg acagacgtcg cggtgagttc aggctttttc atatctcatt gccccccggg 18960
atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt cagcgtgtcc 19020
tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat agtgggattg 19080
tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt 19140
tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact 19200
gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt 19260
gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc 19320
gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga 19380
gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca tgttatcaca 19440
tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt 19500
gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga tagcctttcc 19560
tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc ttttgatgaa 19620
gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc tttgttgaaa 19680
agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg agtagacgag 19740
agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa tacgcaaacc gcctctcccc 19800
gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 19860
agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 19920
tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 19980
aacagctatg accatgatta cgaattcgag ctcggtacca actataacgg tcctaaggta 20040
gcgaaggatc cgctcgctac cttaagagag gatatccctc catcctataa tgtaggctat 20100
aggaactagg gcaaggccgg ccatgcggcc gcaagctggg tgcagcgtga cccggtcgtg 20160
cccctctcta gagataatga gcattgcatg tctaagttat aaaaaattac cacatatttt 20220
ttttgtcaca cttgtttgaa gtgcagttta tctatcttta tacatatatt taaactttac 20280
tctacgaata atataatcta tagtactaca ataatatcag tgttttagag aatcatataa 20340
atgaacagtt agacatggtc taaaggacaa ttgagtattt tgacaacagg actctacagt 20400
tttatctttt tagtgtgcat gtgttctcct ttttttttgc aaatagcttc acctatataa 20460
tacttcatcc attttattag tacatccatt tagggtttag ggttaatggt ttttatagac 20520
taattttttt agtacatcta ttttattcta ttttagcctc taaattaaga aaactaaaac 20580
tctattttag tttttttatt taataattta gatataaaat agaataaaat aaagtgacta 20640
aaaattaaac aaataccctt taagaaatta aaaaaactaa ggaaacattt ttcttgtttc 20700
gagtagataa tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga 20760
accagcagcg tcgcgtcggg ccaagcgaag cagacggcac ggcatctctg tcgctgcctc 20820
tggacccctc tcgagagttc cgctccaccg ttggacttgc tccgctgtcg gcatccagaa 20880
attgcgtggc ggagcggcag acgtgagccg gcacggcagg cggcctcctc ctcctctcac 20940
ggcacggcag ctacggggga ttcctttccc accgctcctt cgctttccct tcctcgcccg 21000
ccgtaataaa tagacacccc ctccacaccc tctttcccca acctcgtgtt gttcggagcg 21060
cacacacaca caaccagatc tcccccaaat ccacccgtcg gcacctccgc ttcaaggtac 21120
gccgctcgtc ctcccccccc ccccctctct accttctcta gatcggcgtt ccggtccatg 21180
gttagggccc ggtagttcta cttctgttca tgtttgtgtt agatccgtgt ttgtgttaga 21240
tccgtgctgc tagcgttcgt acacggatgc gacctgtacg tcagacacgt tctgattgct 21300
aacttgccag tgtttctctt tggggaatcc tgggatggct ctagccgttc cgcagacggg 21360
atcgatttca tgattttttt tgtttcgttg catagggttt ggtttgccct tttcctttat 21420
ttcaatatat gccgtgcact tgtttgtcgg gtcatctttt catgcttttt tttgtcttgg 21480
ttgtgatgat gtggtctggt tgggcggtcg ttctagatcg gagtagaatt ctgtttcaaa 21540
ctacctggtg gatttattaa ttttggatct gtatgtgtgt gccatacata ttcatagtta 21600
cgaattgaag atgatggatg gaaatatcga tctaggatag gtatacatgt tgatgcgggt 21660
tttactgatg catatacaga gatgcttttt gttcgcttgg ttgtgatgat gtggtgtggt 21720
tgggcggtcg ttcattcgtt ctagatcgga gtagaatact gtttcaaact acctggtgta 21780
tttattaatt ttggaactgt atgtgtgtgt catacatctt catagttacg agtttaagat 21840
ggatggaaat atcgatctag gataggtata catgttgatg tgggttttac tgatgcatat 21900
acatgatggc atatgcagca tctattcata tgctctaacc ttgagtacct atctattata 21960
ataaacaagt atgttttata attattttga tcttgatata cttggatgat ggcatatgca 22020
gcagctatat gtggattttt ttagccctgc cttcatacgc tatttatttg cttggtactg 22080
tttcttttgt cgatgctcac cctgttgttt ggtgttactt ctgcagatgg ctcctaagaa 22140
gaagcggaag gttggtattc acggggtgcc tgcggctgac aagaagtact ccatcggcct 22200
cgacatcggc accaacagcg tcggctgggc ggtgatcacc gacgagtaca aggtcccgtc 22260
caagaagttc aaggtcctgg gcaacaccga ccgccactcc atcaagaaga acctcatcgg 22320
cgccctcctc ttcgactccg gcgagacggc ggaggcgacc cgcctcaagc gcaccgcccg 22380
ccgccgctac acccgccgca agaaccgcat ctgctacctc caggagatct tctccaacga 22440
gatggcgaag gtcgacgact ccttcttcca ccgcctcgag gagtccttcc tcgtggagga 22500
ggacaagaag cacgagcgcc accccatctt cggcaacatc gtcgacgagg tcgcctacca 22560
cgagaagtac cccactatct accaccttcg taagaagctt gttgactcta ctgataaggc 22620
tgatcttcgt ctcatctacc ttgctctcgc tcacatgatc aagttccgtg gtcacttcct 22680
tatcgagggt gaccttaacc ctgataactc cgacgtggac aagctcttca tccagctcgt 22740
ccagacctac aaccagctct tcgaggagaa ccctatcaac gcttccggtg tcgacgctaa 22800
ggcgatcctt tccgctaggc tctccaagtc caggcgtctc gagaacctca tcgcccagct 22860
ccctggtgag aagaagaacg gtcttttcgg taacctcatc gctctctccc tcggtctgac 22920
ccctaacttc aagtccaact tcgacctcgc tgaggacgct aagcttcagc tctccaagga 22980
tacctacgac gatgatctcg acaacctcct cgctcagatt ggagatcagt acgctgatct 23040
cttccttgct gctaagaacc tctccgatgc tatcctcctt tcggatatcc ttagggttaa 23100
cactgagatc actaaggctc ctctttctgc ttccatgatc aagcgctacg acgagcacca 23160
ccaggacctc accctcctca aggctcttgt tcgtcagcag ctccccgaga agtacaagga 23220
gatcttcttc gaccagtcca agaacggcta cgccggttac attgacggtg gagctagcca 23280
ggaggagttc tacaagttca tcaagccaat ccttgagaag atggatggta ctgaggagct 23340
tctcgttaag cttaaccgtg aggacctcct taggaagcag aggactttcg ataacggctc 23400
tatccctcac cagatccacc ttggtgagct tcacgccatc cttcgtaggc aggaggactt 23460
ctaccctttc ctcaaggaca accgtgagaa gatcgagaag atccttactt tccgtattcc 23520
ttactacgtt ggtcctcttg ctcgtggtaa ctcccgtttc gcttggatga ctaggaagtc 23580
cgaggagact atcacccctt ggaacttcga ggaggttgtt gacaagggtg cttccgccca 23640
gtccttcatc gagcgcatga ccaacttcga caagaacctc cccaacgaga aggtcctccc 23700
caagcactcc ctcctctacg agtacttcac ggtctacaac gagctcacca aggtcaagta 23760
cgtcaccgag ggtatgcgca agcctgcctt cctctccggc gagcagaaga aggctatcgt 23820
tgacctcctc ttcaagacca accgcaaggt caccgtcaag cagctcaagg aggactactt 23880
caagaagatc gagtgcttcg actccgtcga gatcagcggc gttgaggacc gtttcaacgc 23940
ttctctcggt acctaccacg atctcctcaa gatcatcaag gacaaggact tcctcgacaa 24000
cgaggagaac gaggacatcc tcgaggacat cgtcctcact cttactctct tcgaggatag 24060
ggagatgatc gaggagaggc tcaagactta cgctcatctc ttcgatgaca aggttatgaa 24120
gcagctcaag cgtcgccgtt acaccggttg gggtaggctc tcccgcaagc tcatcaacgg 24180
tatcagggat aagcagagcg gcaagactat cctcgacttc ctcaagtctg atggtttcgc 24240
taacaggaac ttcatgcagc tcatccacga tgactctctt accttcaagg aggatattca 24300
gaaggctcag gtgtccggtc agggcgactc tctccacgag cacattgcta accttgctgg 24360
ttcccctgct atcaagaagg gcatccttca gactgttaag gttgtcgatg agcttgtcaa 24420
ggttatgggt cgtcacaagc ctgagaacat cgtcatcgag atggctcgtg agaaccagac 24480
tacccagaag ggtcagaaga actcgaggga gcgcatgaag aggattgagg agggtatcaa 24540
ggagcttggt tctcagatcc ttaaggagca ccctgtcgag aacacccagc tccagaacga 24600
gaagctctac ctctactacc tccagaacgg tagggatatg tacgttgacc aggagctcga 24660
catcaacagg ctttctgact acgacgtcga ccacattgtt cctcagtctt tccttaagga 24720
tgactccatc gacaacaagg tcctcacgag gtccgacaag aacaggggta agtcggacaa 24780
cgtcccttcc gaggaggttg tcaagaagat gaagaactac tggaggcagc ttctcaacgc 24840
taagctcatt acccagagga agttcgacaa cctcacgaag gctgagaggg gtggcctttc 24900
cgagcttgac aaggctggtt tcatcaagag gcagcttgtt gagacgaggc agattaccaa 24960
gcacgttgct cagatcctcg attctaggat gaacaccaag tacgacgaga acgacaagct 25020
catccgcgag gtcaaggtga tcaccctcaa gtccaagctc gtctccgact tccgcaagga 25080
cttccagttc tacaaggtcc gcgagatcaa caactaccac cacgctcacg atgcttacct 25140
taacgctgtc gttggtaccg ctcttatcaa gaagtaccct aagcttgagt ccgagttcgt 25200
ctacggtgac tacaaggtct acgacgttcg taagatgatc gccaagtccg agcaggagat 25260
cggcaaggcc accgccaagt acttcttcta ctccaacatc atgaacttct tcaagaccga 25320
gatcaccctc gccaacggcg agatccgcaa gcgccctctt atcgagacga acggtgagac 25380
tggtgagatc gtttgggaca agggtcgcga cttcgctact gttcgcaagg tcctttctat 25440
gcctcaggtt aacatcgtca agaagaccga ggtccagacc ggtggcttct ccaaggagtc 25500
tatccttcca aagagaaact cggacaagct catcgctagg aagaaggatt gggaccctaa 25560
gaagtacggt ggtttcgact cccctactgt cgcctactcc gtcctcgtgg tcgccaaggt 25620
ggagaagggt aagtcgaaga agctcaagtc cgtcaaggag ctcctcggca tcaccatcat 25680
ggagcgctcc tccttcgaga agaacccgat cgacttcctc gaggccaagg gctacaagga 25740
ggtcaagaag gacctcatca tcaagctccc caagtactct cttttcgagc tcgagaacgg 25800
tcgtaagagg atgctggctt ccgctggtga gctccagaag ggtaacgagc ttgctcttcc 25860
ttccaagtac gtgaacttcc tctacctcgc ctcccactac gagaagctca agggttcccc 25920
tgaggataac gagcagaagc agctcttcgt ggagcagcac aagcactacc tcgacgagat 25980
catcgagcag atctccgagt tctccaagcg cgtcatcctc gctgacgcta acctcgacaa 26040
ggtcctctcc gcctacaaca agcaccgcga caagcccatc cgcgagcagg ccgagaacat 26100
catccacctc ttcacgctca cgaacctcgg cgcccctgct gctttcaagt acttcgacac 26160
caccatcgac aggaagcgtt acacgtccac caaggaggtt ctcgacgcta ctctcatcca 26220
ccagtccatc accggtcttt acgagactcg tatcgacctt tcccagcttg gtggtgataa 26280
gcgtcctgct gccaccaaaa aggccggaca ggctaagaaa aagaagtagg atcctcccga 26340
tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 26400
gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 26460
gacgttattt atgaggtggg tttttatgat tagagtcccg caattataca tttaatacgc 26520
gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 26580
gttactagat cgggagcacc ggtaaggcgc gccgtagtgc tcgactagta tggaatcggc 26640
agcaaaggag aaatctcaaa attccggcag aacaattttg aatctcgatc cgtagaaacg 26700
agacggtcat tgttttagtt ccaccacgat tatatttgaa atttacgtga gtgtgagtga 26760
gacttgcata agaaaataaa atctttagtt gggaaaaaat tcaataatat aaatgggctt 26820
gagaaggaag cgagggatag gcctttttct aaaataggcc catttaagct attaacaatc 26880
ttcaaaagta ccacagcgct taggtaaaga aagcagctga gtttatatat ggttagagac 26940
gaagtagtga ttgtacactg ctgcaccaga ccgttttaga gctagaaata gcaagttaaa 27000
ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttcaaga 27060
gcttggagtg gatggaccct gacactggaa tcggcagcaa aggaaaatat cagagatctc 27120
ttacagttag tttcgttctt aatccaaact actgcagcct gacagacaaa tgaggatgca 27180
aacaatttta aagtttatct aacgctagct gttttgtttc ttctctctgg tgcaccaacg 27240
acggcgtttt ctcaatcata aagaggcttg ttttacttaa ggccaataat gttgatggat 27300
cgaaagaaga gggcttttaa taaacgagcc cgtttaagct gtaaacgatg tcaaaaacat 27360
cccacatcgt tcagttgaaa atagtagctc tgtttatata ttggtagagt cgactaagag 27420
attgcacagc aggtttcgga ggcgttttag agctagaaat agcaagttaa aataaggcta 27480
gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttcaag agcttggagt 27540
ggatggagtg aagactttgg aatcggcagc aaaggattta ctttaaattt tttcttatgc 27600
agcctgtgat ggataactga atcaaacaaa tggcgtctgg gtttaagaag atctgttttg 27660
gctatgttgg acgaaacaag tgaactttta ggatcaactt cagtttatat atggagctta 27720
tatcgagcaa taagataagt gggcttttta tgtaatttaa tgggctatcg tccatagatt 27780
cactaatacc catgcccagt acccatgtat gcgtttcata taagctccta atttctccca 27840
catcgctcaa atctaaacaa atcttgttgt atatataaca ctgagggagc aacattggtc 27900
attatcggag ctggccctgc gttttagagc tagaaatagc aagttaaaat aaggctagtc 27960
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttcaagagc ttggagtgga 28020
tggaaaggac tacatggaat cggcagcaaa ggagaaatct caaaattccg gcagaacaat 28080
tttgaatctc gatccgtaga aacgagacgg tcattgtttt agttccacca cgattatatt 28140
tgaaatttac gtgagtgtga gtgagacttg cataagaaaa taaaatcttt agttgggaaa 28200
aaattcaata atataaatgg gcttgagaag gaagcgaggg ataggccttt ttctaaaata 28260
ggcccattta agctattaac aatcttcaaa agtaccacag cgcttaggta aagaaagcag 28320
ctgagtttat atatggttag agacgaagta gtgattgccg cggagtcagc taaattgttt 28380
tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 28440
ccgagtcggt gctttttttc aagagcttgg agtggatgga tacgcgtcgg tatcattggc 28500
gcgcctctcg agctagcggc cgcatgcatc gatctcctac atcgtataaa ttagcctata 28560
cgaagttatt gcatctatgt cgggtgcgga gaaagaggta atgaaatggc agtattagat 28620
ctgataactt cgtataatgt atgctatacg aagttatgac tgcaggtcga cacccataat 28680
agctgtttgc caagcttggc actggccgtc gtttttacaa cgtcgtgact gggaaaaccc 28740
tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 28800
cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatgcta 28860
gagcagcttg agcttggatc agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt 28920
tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga ataacggata 28980
tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg ccaaccacag 29040
ggttcccctc gggatcaaag tactttgatc caacccctcc gctgctatag tgcagtcggc 29100
ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt cgcacaagtc ctaagttacg 29160
cgacaggctg ccgccctgcc cttttcctgg cgttttcttg tcgcgtgttt tagtcgcata 29220
aagtagaata cttgcgacta gaaccggaga cattacgcca tgaacaagag cgccgccgct 29280
ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg acttgaccaa ccaacgggcc 29340
gaactgcacg cggccggctg caccaagctg ttttccgaga agatcaccgg caccaggcgc 29400
gaccgcccgg agctggccag gatgcttgac cacctacgcc ctggcgacgt tgtgacagtg 29460
accaggctag accgcctggc ccgcagcacc cgcgacctac tggacattgc cgagcgcatc 29520
caggaggccg gcgcgggcct gcgtagcctg gcagagccgt gggccgacac caccacgccg 29580
gccggccgca tggtgttgac cgtgttcgcc ggcattgccg agttcgagcg ttccctaatc 29640
atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc gaggcgtgaa gtttggcccc 29700
cgccctaccc tcaccccggc acagatcgcg cacgcccgcg agctgatcga ccaggaaggc 29760
cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc gctcgaccct gtaccgcgca 29820
cttgagcgca gcgaggaagt gacgcccacc gaggccaggc ggcgcggtgc cttccgtgag 29880
gacgcattga ccgaggccga cgccctggcg gccgccgaga atgaacgcca agaggaacaa 29940
gcatgaaacc gcaccaggac ggccaggacg aaccgttttt cattaccgaa gagatcgagg 30000
cggagatgat cgcggccggg tacgtgttcg agccgcccgc gcacgtctca accgtgcggc 30060
tgcatgaaat cctggccggt ttgtctgatg ccaagctggc ggcctggccg gccagcttgg 30120
ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag taaaacagct 30180
tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata cgcaagggga 30240
acgcatgaag gttatcgctg tacttaacca gaaaggcggg tcaggcaaga cgaccatcgc 30300
aacccatcta gcccgcgccc tgcaactcgc cggggccgat gttctgttag tcgattccga 30360
tccccagggc agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc taaccgttgt 30420
cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc gcgacttcgt 30480
agtgatcgac ggagcgcccc aggcggcgga cttggctgtg tccgcgatca aggcagccga 30540
cttcgtgctg attccggtgc agccaagccc ttacgacata tgggccaccg ccgacctggt 30600
ggagctggtt aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg cctttgtcgt 30660
gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc tggccgggta 30720
cgagctgccc attcttgagt cccgtatcac gcagcgcgtg agctacccag gcactgccgc 30780
cgccggcaca accgttcttg aatcagaacc cgagggcgac gctgcccgcg aggtccaggc 30840
gctggccgct gaaattaaat caaaactcat ttgagttaat gaggtaaaga gaaaatgagc 30900
aaaagcacaa acacgctaag tgccggccgt ccgagcgcac gcagcagcaa ggctgcaacg 30960
ttggccagcc tggcagacac gccagccatg aagcgggtca actttcagtt gccggcggag 31020
gatcacacca agctgaagat gtacgcggta cgccaaggca agaccattac cgagctgcta 31080
tctgaataca tcgcgcagct accagagtaa atgagcaaat gaataaatga gtagatgaat 31140
tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg acgccgtgga 31200
atgccccatg tgtggaggaa cgggcggttg gccaggcgta agcggctggg ttgtctgccg 31260
gccctgcaat ggcactggaa cccccaagcc cgaggaatcg gcgtgacggt cgcaaaccat 31320
ccggcccggt acaaatcggc gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg 31380
cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag 31440
cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga 31500
ttaggaagcc gcccaagggc gacgagcaac cagatttttt cgttccgatg ctctatgacg 31560
tgggcacccg cgatagtcgc agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg 31620
accgacgagc tggcgaggtg atccgctacg agcttccaga cgggcacgta gaggtttccg 31680
cagggccggc cggcatggcc agtgtgtggg attacgacct ggtactgatg gcggtttccc 31740
atctaaccga atccatgaac cgataccggg aagggaaggg agacaagccc ggccgcgtgt 31800
tccgtccaca cgttgcggac gtactcaagt tctgccggcg agccgatggc ggaaagcaga 31860
aagacgacct ggtagaaacc tgcattcggt taaacaccac gcacgttgcc atgcagcgta 31920
cgaagaaggc caagaacggc cgcctggtga cggtatccga gggtgaagcc ttgattagcc 31980
gctacaagat cgtaaagagc gaaaccgggc ggccggagta catcgagatc gagctagctg 32040
attggatgta ccgcgagatc acagaaggca agaacccgga cgtgctgacg gttcaccccg 32100
attacttttt gatcgatccc ggcatcggcc gttttctcta ccgcctggca cgccgcgccg 32160
caggcaaggc agaagccaga tggttgttca agacgatcta cgaacgcagt ggcagcgccg 32220
gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg 32280
agtacgattt gaaggaggag gcggggcagg ctggcccgat cctagtcatg cgctaccgca 32340
acctgatcga gggcgaagca tccgccggtt cctaatgtac ggagcagatg ctagggcaaa 32400
ttgccctagc aggggaaaaa ggtcgaaaag gtctgtttcc tgtggatagc acgtacattg 32460
ggaacccaaa gccgtacatt gggaaccgga acccgtacat tgggaaccca aagccgtaca 32520
ttgggaaccg gtcacacatg taagtgactg atataaaaga gaaaaaaggc gatttttccg 32580
cctaaaactc tttaaaactt attaaaactc ttaaaacccg cctggcctgt gcataactgt 32640
ctggccagcg cacagccgaa gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc 32700
tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg ccgctcaaaa atggctggcc 32760
tacggccagg caatctacca gggcgcggac aagccgcgcc gtcgccactc gaccgccggc 32820
gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 32880
atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 32940
cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt 33000
agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag 33060
tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc 33120
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 33180
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 33240
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 33300
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 33360
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 33420
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 33480
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 33540
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 33600
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 33660
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 33720
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 33780
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 33840
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 33900
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 33960
tggtcatgca ttctaggtac taaaacaatt catccagtaa aatataatat tttattttct 34020
cccaatcagg cttgatcccc agtaagtcaa aaaatagctc gacatactgt tcttccccga 34080
tatcctccct gatcgaccgg acgcagaagg caatgtcata ccacttgtcc gccctgccgc 34140
ttctcccaag atcaataaag ccacttactt tgccatcttt cacaaagatg ttgctgtctc 34200
ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt ttccgtcttt aaaaaatcat 34260
acagctcgcg cggatcttta aatggagtgt cttcttccca gttttcgcaa tccacatcgg 34320
ccagatcgtt attcagtaag taatccaatt cggctaagcg gctgtctaag ctattcgtat 34380
agggacaatc cgatatgtcg atggagtgaa agagcctgat gcactccgca tacagctcga 34440
taatcttttc agggctttgt tcatcttcat actcttccga gcaaaggacg ccatcggcct 34500
cactcatgag cagattgctc cagccatcat gccgttcaaa gtgcaggacc tttggaacag 34560
gcagctttcc ttccagccat agcatcatgt ccttttcccg ttccacatca taggtggtcc 34620
ctttataccg gctgtccgtc atttttaaat ataggttttc attttctccc accagcttat 34680
ataccttagc aggagacatt ccttccgtat cttttacgca gcggtatttt tcgatcagtt 34740
ttttcaattc cggtgatatt ctcattttag ccatttatta tttccttcct cttttctaca 34800
gtatttaaag ataccccaag aagctaatta taacaagacg aactccaatt cactgttcct 34860
tgcattctaa aaccttaaat accagaaaac agctttttca aagttgtttt caaagttggc 34920
gtataacata gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg cagcaacgct 34980
ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc atccgtgttt caaacccggc 35040
agcttagttg ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg ccgccttaca 35100
acggctctcc cgctgacgcc gtcccggact gatgggctgc ctgtatcgag tggtgatttt 35160
gtgccgagct gccggtcggg gagctgttgg ctggctgg 35198
<210> 10
<211> 22
<212> DNA
<213> U-F引物(人工序列)
<400> 10
ctccgtttta cctgtggaat cg 22
<210> 11
<211> 36
<212> DNA
<213> OL-C11 Rev引物(人工序列)
<400> 11
ggtctggtgc agcagtgtac aatcactact tcgtct 36
<210> 12
<211> 36
<212> DNA
<213> OL-C12 For(人工序列)
<400> 12
tacactgctg caccagaccg ttttagagct agaaat 36
<210> 13
<211> 20
<212> DNA
<213> gR-R引物(人工序列)
<400> 13
cggaggaaaa ttccatccac 20
<210> 14
<211> 42
<212> DNA
<213> Bsa-C1 For引物(人工序列)
<400> 14
ttcagaggtc tctctcgact agtatggaat cggcagcaaa gg 42
<210> 15
<211> 37
<212> DNA
<213> Bsa-C1 Rev引物(人工序列)
<400> 15
agcgtgggtc tcgtcagggt ccatccactc caagctc 37
<210> 16
<211> 36
<212> DNA
<213> OL-C21 Rev引物(人工序列)
<400> 16
gcctccgaaa cctgctgtgc aatctcttag tcgact 36
<210> 17
<211> 36
<212> DNA
<213> OL-C22 For引物(人工序列)
<400> 17
cacagcaggt ttcggaggcg ttttagagct agaaat 36
<210> 18
<211> 38
<212> DNA
<213> Bsa-C2 For引物(人工序列)
<400> 18
ttcagaggtc tctctgacac tggaatcggc agcaaagg 38
<210> 19
<211> 38
<212> DNA
<213> Bsa-C2Rev引物(人工序列)
<400> 19
agcgtgggtc tcgtcttcac tccatccact ccaagctc 38
<210> 20
<211> 35
<212> DNA
<213> OL-C31 Rev引物(人工序列)
<400> 20
gcagggccag ctccgataat gaccaatgtt gctcc 35
<210> 21
<211> 36
<212> DNA
<213> OL-C32 For引物(人工序列)
<400> 21
ttatcggagc tggccctgcg ttttagagct agaaat 36
<210> 22
<211> 38
<212> DNA
<213> Bsa-C3 For引物(人工序列)
<400> 22
ttcagaggtc tctaagactt tggaatcggc agcaaagg 38
<210> 23
<211> 38
<212> DNA
<213> Bsa-C3Rev引物(人工序列)
<400> 23
agcgtgggtc tcgagtcctt tccatccact ccaagctc 38
<210> 24
<211> 36
<212> DNA
<213> OL-C41 Rev引物(人工序列)
<400> 24
aatttagctg actccgcggc aatcactact tcgtct 36
<210> 25
<211> 36
<212> DNA
<213> OL-C42 For引物(人工序列)
<400> 25
ccgcggagtc agctaaattg ttttagagct agaaat 36
<210> 26
<211> 38
<212> DNA
<213> Bsa-C4 For引物(人工序列)
<400> 26
ttcagaggtc tctgactaca tggaatcggc agcaaagg 38
<210> 27
<211> 42
<212> DNA
<213> Bsa-C4Rev引物(人工序列)
<400> 27
agcgtgggtc tcgaccgacg cgtatccatc cactccaagc tc 42
<210> 29
<211> 3146
<212> DNA
<213> pYLsgRNA-AtU6-1质粒(人工序列)
<400> 29
ggatccagcg tgggtctcgg ttttagagct agaaatagca agttaaaata aggctagtcc 60
gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat 120
ggaattttcc tccgttttac ctgtggaatc ggcagcaaag gagaaatctc aaaattccgg 180
cagaacaatt ttgaatctcg atccgtagaa acgagacggt cattgtttta gttccaccac 240
gattatattt gaaatttacg tgagtgtgag tgagacttgc ataagaaaat aaaatcttta 300
gttgggaaaa aattcaataa tataaatggg cttgagaagg aagcgaggga taggcctttt 360
tctaaaatag gcccatttaa gctattaaca atcttcaaaa gtaccacagc gcttaggtaa 420
agaaagcagc tgagtttata tatggttaga gacgaagtag tgattgagag acctctgaag 480
ataacatact aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 540
gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 600
aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 660
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 720
tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 780
ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 840
tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 900
agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 960
cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 1020
tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 1080
gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 1140
cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 1200
atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 1260
agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 1320
gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 1380
ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 1440
cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 1500
ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 1560
atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 1620
gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 1680
tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 1740
gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 1800
gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 1860
tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 1920
ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 1980
tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 2040
ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 2100
ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 2160
tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 2220
ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 2280
tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 2340
tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 2400
actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 2460
cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 2520
gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 2580
tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 2640
ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 2700
ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 2760
cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 2820
cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 2880
gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 2940
attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 3000
ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 3060
gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 3120
attacgaatt cgagctcggt acccgg 3146
<210> 29
<211> 3162
<212> DNA
<213> pYLsgRNA-AtU6-29质粒(人工序列)
<400> 29
ggatccagcg tgggtctcgg ttttagagct agaaatagca agttaaaata aggctagtcc 60
gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat 120
ggaattttcc tccgttttac ctgtggaatc ggcagcaaag gaaaatatca gagatctctt 180
acagttagtt tcgttcttaa tccaaactac tgcagcctga cagacaaatg aggatgcaaa 240
caattttaaa gtttatctaa cgctagctgt tttgtttctt ctctctggtg caccaacgac 300
ggcgttttct caatcataaa gaggcttgtt ttacttaagg ccaataatgt tgatggatcg 360
aaagaagagg gcttttaata aacgagcccg tttaagctgt aaacgatgtc aaaaacatcc 420
cacatcgttc agttgaaaat agtagctctg tttatatatt ggtagagtcg actaagagat 480
tgagagacct ctgaagataa catactaagc ttggcactgg ccgtcgtttt acaacgtcgt 540
gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 600
agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 660
aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 720
cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga 780
cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 840
agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 900
aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 960
ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 1020
tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 1080
atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 1140
attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 1200
gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 1260
agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 1320
aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 1380
cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 1440
cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 1500
actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 1560
cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 1620
ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 1680
ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 1740
gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 1800
gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 1860
ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 1920
cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 1980
caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 2040
taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 2100
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 2160
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 2220
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 2280
aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 2340
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 2400
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 2460
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 2520
ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 2580
ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 2640
tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 2700
tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 2760
ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 2820
gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 2880
cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 2940
gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 3000
agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 3060
tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 3120
aacagctatg accatgatta cgaattcgag ctcggtaccc gg 3162
<210> 30
<211> 3146
<212> DNA
<213> pYLsgRNA-AtU3b质粒(人工序列)
<400> 30
ggatccagcg tgggtctcgg ttttagagct agaaatagca agttaaaata aggctagtcc 60
gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat 120
ggaattttcc tccgttttac ctgtggaatc ggcagcaaag gacgcgttga cattgtagga 180
ctatattgct ctaataaagg aggcagctat gctggccgtc gttttacaac gtcgtgactg 240
ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 300
gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 360
ctaaataagc ttatgatttc ttttttctta cgaattttgc gtcccacatc ggtaagcgag 420
tgaagaaata actgctttat atatggctac aaagcaccat tggtcaagag acctctgaag 480
ataacatact aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 540
gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 600
aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 660
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 720
tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 780
ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 840
tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 900
agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 960
cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 1020
tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 1080
gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 1140
cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 1200
atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 1260
agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 1320
gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 1380
ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 1440
cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 1500
ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 1560
atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 1620
gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 1680
tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 1740
gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 1800
gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 1860
tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 1920
ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 1980
tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 2040
ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 2100
ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 2160
tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 2220
ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 2280
tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 2340
tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 2400
actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 2460
cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 2520
gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 2580
tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 2640
ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 2700
ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 2760
cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 2820
cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 2880
gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 2940
attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 3000
ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 3060
gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 3120
attacgaatt cgagctcggt acccgg 3146
<210> 31
<211> 22
<212> DNA
<213> Pubi-F1引物(人工序列)
<400> 31
gcggtgtcat ctatgttact ag 22
<210> 32
<211> 23
<212> DNA
<213> SP-R引物(人工序列)
<400> 32
cccgacatag atgcaataac ttc 23
<210> 33
<211> 2018
<212> DNA
<213> pSL-Pubi-H-4-4(人工序列)
<400> 33
gcggtgtcat ctatgttact agatcgggag caccggtaag gcgcgccgta gtgctcgact 60
agtatggaat cggcagcaaa ggagaaatct caaaattccg gcagaacaat tttgaatctc 120
gatccgtaga aacgagacgg tcattgtttt agttccacca cgattatatt tgaaatttac 180
gtgagtgtga gtgagacttg cataagaaaa taaaatcttt agttgggaaa aaattcaata 240
atataaatgg gcttgagaag gaagcgaggg ataggccttt ttctaaaata ggcccattta 300
agctattaac aatcttcaaa agtaccacag cgcttaggta aagaaagcag ctgagtttat 360
atatggttag agacgaagta gtgattgtac actgctgcac cagaccgttt tagagctaga 420
aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 480
gctttttttc aagagcttgg agtggatgga ccctgacact ggaatcggca gcaaaggaaa 540
atatcagaga tctcttacag ttagtttcgt tcttaatcca aactactgca gcctgacaga 600
caaatgagga tgcaaacaat tttaaagttt atctaacgct agctgttttg tttcttctct 660
ctggtgcacc aacgacggcg ttttctcaat cataaagagg cttgttttac ttaaggccaa 720
taatgttgat ggatcgaaag aagagggctt ttaataaacg agcccgttta agctgtaaac 780
gatgtcaaaa acatcccaca tcgttcagtt gaaaatagta gctctgttta tatattggta 840
gagtcgacta agagattgca cagcaggttt cggaggcgtt ttagagctag aaatagcaag 900
ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 960
caagagcttg gagtggatgg agtgaagact ttggaatcgg cagcaaagga tttactttaa 1020
attttttctt atgcagcctg tgatggataa ctgaatcaaa caaatggcgt ctgggtttaa 1080
gaagatctgt tttggctatg ttggacgaaa caagtgaact tttaggatca acttcagttt 1140
atatatggag cttatatcga gcaataagat aagtgggctt tttatgtaat ttaatgggct 1200
atcgtccata gattcactaa tacccatgcc cagtacccat gtatgcgttt catataagct 1260
cctaatttct cccacatcgc tcaaatctaa acaaatcttg ttgtatatat aacactgagg 1320
gagcaacatt ggtcattatc ggagctggcc ctgcgtttta gagctagaaa tagcaagtta 1380
aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttcaa 1440
gagcttggag tggatggaaa ggactacatg gaatcggcag caaaggagaa atctcaaaat 1500
tccggcagaa caattttgaa tctcgatccg tagaaacgag acggtcattg ttttagttcc 1560
accacgatta tatttgaaat ttacgtgagt gtgagtgaga cttgcataag aaaataaaat 1620
ctttagttgg gaaaaaattc aataatataa atgggcttga gaaggaagcg agggataggc 1680
ctttttctaa aataggccca tttaagctat taacaatctt caaaagtacc acagcgctta 1740
ggtaaagaaa gcagctgagt ttatatatgg ttagagacga agtagtgatt gccgcggagt 1800
cagctaaatt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac 1860
ttgaaaaagt ggcaccgagt cggtgctttt tttcaagagc ttggagtgga tggatacgcg 1920
tcggtatcat tggcgcgcct ctcgagctag cggccgcatg catcgatctc ctacatcgta 1980
taaattagcc tatacgaagt tattgcatct atgtcggg 2018

Claims (6)

1.一种通过基因编辑技术提高番茄中番茄红素含量的方法，包括以下步骤:
1)分别构建Lcyb1、Lcyb2、CycB和LcyE基因的靶序列T1～T4，然后通过靶序列分别构建其对应的T1sgRNA～T4sgRNA表达盒；
2)将步骤1)中的T1sgRNA～T4sgRNA表达盒与crispr/cas9表达载体进行组装，得到重组质粒；
3)利用农杆菌侵染法将步骤2)中的重组质粒转化到番茄中，得到番茄新材料，因为新番茄材料中与番茄红素环化酶合成相关基因会存在一个或多个基因的突变，从而抑制番茄红素环化酶的合成，使得新番茄材料的番茄红素含量提高；
其中：T1～T4的核苷酸序列如序列1～4所示。
2.根据权利要求1所述的通过基因编辑技术提高番茄中番茄红素含量的方法，其特征在于，所述步骤1)中，T1sgRNA表达盒中含有T1sgRNA序列如序列5所示；T2sgRNA表达盒中含有T2sgRNA序列如序列6所示；T3sgRNA表达盒中含有T3sgRNA序列如序列7所示；T4sgRNA表达盒中含有T4sgRNA序列如序列8所示。
3.根据权利要求1所述的通过基因编辑技术提高番茄中番茄红素含量的方法，其特征在于，所述步骤2)中所述的重组质粒的核苷酸序列如序列9所示。
4.根据权利要求1中所述的靶序列T1～T4在制备高番茄红素的番茄材料中的应用。
5.根据权利要求1或2所述的T1sgRNA～T4sgRNA序列在制备高番茄红素的番茄材料中的应用。
6.根据权利要求1或3所述的重组载体在制备高番茄红素的番茄材料中的应用。
CN201910899703.5A 2019-09-23 2019-09-23 一种通过基因编辑技术提高番茄中番茄红素含量的方法 Pending CN110499319A (zh)

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CN104531755A (zh) * 2014-12-19 2015-04-22 西南大学 干扰lcyb、lcye表达同时异位表达tt8在制备红色花瓣芸薹属植物中的应用
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