柚（citrus grandis Osbeck）单染色体AFLP分子标记体系及文库的建立
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摘要
柚是我国重要的水果。柚遗传背景较复杂,生产周期长,一直以来难以建立精确的物理图谱,这为进行柚基因组学的研究、分子标记辅助育种以及克隆一些重要的园艺性状基因造成了困难,染色体微分离和微克隆技术的应用为柚类遗传学的研究提供了新的思路。针对柚单染色体极小,形态辨别和微分离困难,本试验采用随机微分离的方法,通过LA-PCR-AFLP技术对染色体进行同源划分,并使用Southern杂交技术对单染色体微克隆质量和同源划分的正确性进行鉴定和验证。最后将各同源单染色体文库合并,能大大提高同源染色体文库的覆盖度,如果将各类同源染色体文库合并,则建立了整个柚染色体文库。
1、对柚染色体的制片和微分离技术进行了研究。比较了压片-液氮速冻揭片法和悬液法制片的优劣；优化了纤维素果胶酶的浓度比例和酶解时间：2%纤维素酶和1%果胶酶,酶解1 h；比较了卡宝品红和铁钒苏木精两种染色方法,并最终选用了卡宝品红染色法；对玻璃针的制备、分离方法进行了选择和改进。良好的制片效果大大提高了单染色体显微分离的质量和效率。
2、针对柚单染色体微克隆受外源污染干扰严重的情况,通过对比试验,对柚单染色体LA-PCR-AFLP技术体系中外源DNA污染问题中不同试验用水、加接头连接前后步骤对水污染的敏感度以及LA-PCR形成的非特异性亮带现象进行了研究。结果表明：8种试验用水中BT水(Bioteke, RNase-free and DNase-free Water)最佳；水的质量对LA-PCR过程中接头连接之前的步骤影响最大,对加接头之后的步骤也有一定的影响；非特异性亮带为引物自连形成的多聚体,与外源污染无关。同时试验了Southern杂交对柚单染色体LA-PCR过程中外源污染程度的检测,及细胞质污染的影响等。本试验通过对外源污染的来源和关键步骤实施严格防控,经过dot blot验证,使外源污染能够得到有效控制。这为建立高质量的柚单染色体文库奠定了基础。
3、针对柚叶片多糖的情况,通过3种基因组DNA提取方法比较,最终选用SDS-KAC法并获得了较高质量的柚基因组DNA；对酶切时间和ATP浓度对连接反应的影响进行了研究,证明1-5 h均能满足单染色体的酶切需要,但是为保证不同单染色体扩增结果的一致性,最后选择较长的酶切时间4-5 h；ATP连接终浓度在大于5mM的时候可能抑制连接反应,而在制接头的时候不额外添加ATP也不会对实验结果造成太大影响；显微分离了166个柚单染色体,并通过dot-blot杂交和Southern印迹杂交进行了验证,从中筛选出信号较强的55个单染色体用于进一步试验。对柚单染色体LA-PCR-AFLP技术体系中的关键参数进行了优化,结果表明：退火温度较高的程序TD3(退火梯度：72℃→65℃30 s(-0.5℃/cyc))扩增效果较好；稀释100倍(10 ng)为最适模板量；筛选出了扩增多态性较好的10组引物对；最终建立了5张不同引物对组合扩增的柚不同单染色体的AFLP-SDS-PAGE指纹图谱。
4、获得了高质量的官溪蜜柚总RNA,逆转录合成双链cDNA,经LA-PCR过程,获得cDNA扩增产物。获得的产物经纯化后与pMD18-T-vector载体连接,进行T-A克隆,获得大量cDNA文库片段的克隆子。从其中随机选择134个克隆子经纯化后用于后续试验。
5、建立了适用于柚单染色体同源鉴定的dot-blot杂交体系：1)探针制备时DIG-High Prime (vial 1)的用量可减少为2.5μL；2)单染色体严格纯化后制作探针,探针制成后不用再纯化；3)使用柚单染色体第一轮LA-PCR产物制作探针效果较好；4)柚单染色体差异片段和柚cDNA扩增文库片段必须经纯化后作为模板；5)当使用柚单染色体探针进行dot-blot杂交时,柚单染色体文库、差异片段、柚cDNA扩增文库片段模板的上样量应小于1 ng。
6、对5对引物扩增的AFLP-SDS-PAGE指纹图谱多态性片段的聚类分析,初步把55条单染色体体划分为10类；将这10类染色体分别与134个cDNA扩增文库克隆子进行dot-blot杂交,验证了不同单染色体筛选cDNA克隆文库进行同源性划分的可行性。通过进一步的Southern杂交验证,确保了同源划分的正确性。
本试验对柚单染色体进行了初步的同源划分和建立了同源染色体文库,并经过Southern杂交验证和鉴定。但染色体文库的完整性和质量可能仍然不够高,需要不断的进行修弥补和修正。若要建立比较完整和高质量的文库，还要做大量的补充和验证工作。
单染色体文库的建立为以后建立柚精确物理图谱奠定了基础,对于进一步进行柚基因组学和遗传学研究具有重要的理论和现实意义。同时为柚分子遗传育种和优良基因的定位和克隆提供了更加方便、准确、快捷的方法。
Pomelo is an important fruit in China. Beacause of its complex genetic background and long growth cycle, it's hard to construct its accurate physical map., which causes difficulty in the stuys of genomics, molecular marker-assisted breeding and cloning of genes with important horticultural characters.Chromosome microdissection and microcloning technique provides new thoughts of the genetic research of Pomelo. We used the method of random single chromosome microdissection in view of the most small of the single chromosome of Pomelo and hard to recognize. The LA-PCR-AFLP technique was used in our study to classify the chromosomes of Pomelo. And the Southern blot was used to identify the results. The single chromosomes which are homologous were collected together in the end, so as to construct the entire chromosome library of Pomelo.
1、Made a study in the chromosome preparation and microdissection of Pomelo, compared two overslip-removing methods between quick-froze by liquid nitrogen and suspension. Optimized the concentration ratio between cellulase and pectinase, and their enzyme digestion time:2% cellulase and 1% pectinase, and digested 1 h; compared two dyeing methods between Carbor fuchsin and Hematoxylin, and finally selected the Carbor fuchsin method; Chose and improved on the glass needle preparing and ways of microdissection. A favorable preparation improved more on the quality and efficiency in microdissection of single chromosome.
2、With controlled trials, exogenous DNA contaminations in the LA-PCR-AFLP technical system of the single chromosome in Pcmelo(Citrus grandis Osbeck) were studied using of different types of water, sensitivity test to contaminations of the water between the fore-and-after steps of adding adapter to link and nonspecific bright bands formed in LA-PCR. The results showed that the BT Water(Bioteke, RNase-free and DNase-free Water) was the optimum water among the eight types of test water; The quality of the water had more important influence on the step before adding adapter to link than afterwards; The nonspecific bright bands were primer polymers which had little to do with exogenous contaminations. Exogenous contaminations could be effectively confined by strictly controlling the sources of contamination and strengthening prevention in key steps, and verified by dot blotting. Our research have laid foundation for establishing of high-quality Pomelo single chromosome libraries.
3、Got high-quality genomic DNA of Pomelo extracted by SDS-KAC through comparing 3 extracting methods. And made a study to the time of enzyme digestion and the ATP concentration for ligation:a longer digestion time of 4-5 h was used even the digestion times from 1-5 h were all available in order to keep the consistency; and when ligase concentration was more than 5 mM would stop the reaction, and it had less influence even didn't add extra ATP at ligation. The key parameters of LA-PCR-AFLP Technical system were optimized for available PCR protocol, optimum template concentration and better primer pairs on the base of chromosome microdissection and microcloning technique. Results showed that program TD3 which annealing temperature was higher (72℃→65℃30 s (-0.5℃/ cyc)) had better amplified effect; the optimum template amount was diluted with 100 times(10 ng) and 10 primer pairs had better amplified polymorphism. And finally 5 LA-PCR-AFLP fingerprints of different single chromosomes in pomelo were constructed amplified by different groups of primer pairs.
4、The high-quality total RNA was obtained and reverse transcribed, then got the amplified products of cDNA by LA-PCR. The products were purified and linked with pMD18-T-vector and then transformed to E.coli and got a hundred of clones, which were purified for following studys.
5、Constructed a dot-blot homologous identification system which is suitable for single chromosome of Pomelo:1)Use 2.5μL DIG-High Prime (vial 1) for probes preparation.2)Single chromosomes should be purified for probes preparation, and need not purify when have made.3)Use the first round of PCR products for probes preparation.4)The differential fragments of single chromosomes and the fragments of cDNA clonicons should be purified before used.5)The sample amounts was 1 ng when the single chromosomes of Pomelo and differential segments and cDNA clonicons were used as probe.
6、Made a cluster analysis of the polymorphic segments from AFLP-SDS-PAGE maps amplified by 5 prime pairs, and the 55 single chromosomes were classified to 10 groups. Then the 10 groups were hybridized with 134 clonicons from cDNA library, in order to proving the validity of the clusters, and a further Southern blot ensured the validity.
A preliminary classification was made and the homologous chromosome libraries were preliminarily constructed, but it is not enough in both integrity and quality, great afforts should be maken in case of construction of high-quality and integrity chromosome library.
The results in this study laid the foundation for the construction of accurate physic maps in future, and was very meaningful for subsequent genomic studys in Pomelo. And provided easier, faster and more accurate ways for genetic breeding and the location and cloning of useful genes in Pomelo.
1、对柚染色体的制片和微分离技术进行了研究。比较了压片-液氮速冻揭片法和悬液法制片的优劣；优化了纤维素果胶酶的浓度比例和酶解时间：2%纤维素酶和1%果胶酶,酶解1 h；比较了卡宝品红和铁钒苏木精两种染色方法,并最终选用了卡宝品红染色法；对玻璃针的制备、分离方法进行了选择和改进。良好的制片效果大大提高了单染色体显微分离的质量和效率。
2、针对柚单染色体微克隆受外源污染干扰严重的情况,通过对比试验,对柚单染色体LA-PCR-AFLP技术体系中外源DNA污染问题中不同试验用水、加接头连接前后步骤对水污染的敏感度以及LA-PCR形成的非特异性亮带现象进行了研究。结果表明：8种试验用水中BT水(Bioteke, RNase-free and DNase-free Water)最佳；水的质量对LA-PCR过程中接头连接之前的步骤影响最大,对加接头之后的步骤也有一定的影响；非特异性亮带为引物自连形成的多聚体,与外源污染无关。同时试验了Southern杂交对柚单染色体LA-PCR过程中外源污染程度的检测,及细胞质污染的影响等。本试验通过对外源污染的来源和关键步骤实施严格防控,经过dot blot验证,使外源污染能够得到有效控制。这为建立高质量的柚单染色体文库奠定了基础。
3、针对柚叶片多糖的情况,通过3种基因组DNA提取方法比较,最终选用SDS-KAC法并获得了较高质量的柚基因组DNA；对酶切时间和ATP浓度对连接反应的影响进行了研究,证明1-5 h均能满足单染色体的酶切需要,但是为保证不同单染色体扩增结果的一致性,最后选择较长的酶切时间4-5 h；ATP连接终浓度在大于5mM的时候可能抑制连接反应,而在制接头的时候不额外添加ATP也不会对实验结果造成太大影响；显微分离了166个柚单染色体,并通过dot-blot杂交和Southern印迹杂交进行了验证,从中筛选出信号较强的55个单染色体用于进一步试验。对柚单染色体LA-PCR-AFLP技术体系中的关键参数进行了优化,结果表明：退火温度较高的程序TD3(退火梯度：72℃→65℃30 s(-0.5℃/cyc))扩增效果较好；稀释100倍(10 ng)为最适模板量；筛选出了扩增多态性较好的10组引物对；最终建立了5张不同引物对组合扩增的柚不同单染色体的AFLP-SDS-PAGE指纹图谱。
4、获得了高质量的官溪蜜柚总RNA,逆转录合成双链cDNA,经LA-PCR过程,获得cDNA扩增产物。获得的产物经纯化后与pMD18-T-vector载体连接,进行T-A克隆,获得大量cDNA文库片段的克隆子。从其中随机选择134个克隆子经纯化后用于后续试验。
5、建立了适用于柚单染色体同源鉴定的dot-blot杂交体系：1)探针制备时DIG-High Prime (vial 1)的用量可减少为2.5μL；2)单染色体严格纯化后制作探针,探针制成后不用再纯化；3)使用柚单染色体第一轮LA-PCR产物制作探针效果较好；4)柚单染色体差异片段和柚cDNA扩增文库片段必须经纯化后作为模板；5)当使用柚单染色体探针进行dot-blot杂交时,柚单染色体文库、差异片段、柚cDNA扩增文库片段模板的上样量应小于1 ng。
6、对5对引物扩增的AFLP-SDS-PAGE指纹图谱多态性片段的聚类分析,初步把55条单染色体体划分为10类；将这10类染色体分别与134个cDNA扩增文库克隆子进行dot-blot杂交,验证了不同单染色体筛选cDNA克隆文库进行同源性划分的可行性。通过进一步的Southern杂交验证,确保了同源划分的正确性。
本试验对柚单染色体进行了初步的同源划分和建立了同源染色体文库,并经过Southern杂交验证和鉴定。但染色体文库的完整性和质量可能仍然不够高,需要不断的进行修弥补和修正。若要建立比较完整和高质量的文库，还要做大量的补充和验证工作。
单染色体文库的建立为以后建立柚精确物理图谱奠定了基础,对于进一步进行柚基因组学和遗传学研究具有重要的理论和现实意义。同时为柚分子遗传育种和优良基因的定位和克隆提供了更加方便、准确、快捷的方法。
Pomelo is an important fruit in China. Beacause of its complex genetic background and long growth cycle, it's hard to construct its accurate physical map., which causes difficulty in the stuys of genomics, molecular marker-assisted breeding and cloning of genes with important horticultural characters.Chromosome microdissection and microcloning technique provides new thoughts of the genetic research of Pomelo. We used the method of random single chromosome microdissection in view of the most small of the single chromosome of Pomelo and hard to recognize. The LA-PCR-AFLP technique was used in our study to classify the chromosomes of Pomelo. And the Southern blot was used to identify the results. The single chromosomes which are homologous were collected together in the end, so as to construct the entire chromosome library of Pomelo.
1、Made a study in the chromosome preparation and microdissection of Pomelo, compared two overslip-removing methods between quick-froze by liquid nitrogen and suspension. Optimized the concentration ratio between cellulase and pectinase, and their enzyme digestion time:2% cellulase and 1% pectinase, and digested 1 h; compared two dyeing methods between Carbor fuchsin and Hematoxylin, and finally selected the Carbor fuchsin method; Chose and improved on the glass needle preparing and ways of microdissection. A favorable preparation improved more on the quality and efficiency in microdissection of single chromosome.
2、With controlled trials, exogenous DNA contaminations in the LA-PCR-AFLP technical system of the single chromosome in Pcmelo(Citrus grandis Osbeck) were studied using of different types of water, sensitivity test to contaminations of the water between the fore-and-after steps of adding adapter to link and nonspecific bright bands formed in LA-PCR. The results showed that the BT Water(Bioteke, RNase-free and DNase-free Water) was the optimum water among the eight types of test water; The quality of the water had more important influence on the step before adding adapter to link than afterwards; The nonspecific bright bands were primer polymers which had little to do with exogenous contaminations. Exogenous contaminations could be effectively confined by strictly controlling the sources of contamination and strengthening prevention in key steps, and verified by dot blotting. Our research have laid foundation for establishing of high-quality Pomelo single chromosome libraries.
3、Got high-quality genomic DNA of Pomelo extracted by SDS-KAC through comparing 3 extracting methods. And made a study to the time of enzyme digestion and the ATP concentration for ligation:a longer digestion time of 4-5 h was used even the digestion times from 1-5 h were all available in order to keep the consistency; and when ligase concentration was more than 5 mM would stop the reaction, and it had less influence even didn't add extra ATP at ligation. The key parameters of LA-PCR-AFLP Technical system were optimized for available PCR protocol, optimum template concentration and better primer pairs on the base of chromosome microdissection and microcloning technique. Results showed that program TD3 which annealing temperature was higher (72℃→65℃30 s (-0.5℃/ cyc)) had better amplified effect; the optimum template amount was diluted with 100 times(10 ng) and 10 primer pairs had better amplified polymorphism. And finally 5 LA-PCR-AFLP fingerprints of different single chromosomes in pomelo were constructed amplified by different groups of primer pairs.
4、The high-quality total RNA was obtained and reverse transcribed, then got the amplified products of cDNA by LA-PCR. The products were purified and linked with pMD18-T-vector and then transformed to E.coli and got a hundred of clones, which were purified for following studys.
5、Constructed a dot-blot homologous identification system which is suitable for single chromosome of Pomelo:1)Use 2.5μL DIG-High Prime (vial 1) for probes preparation.2)Single chromosomes should be purified for probes preparation, and need not purify when have made.3)Use the first round of PCR products for probes preparation.4)The differential fragments of single chromosomes and the fragments of cDNA clonicons should be purified before used.5)The sample amounts was 1 ng when the single chromosomes of Pomelo and differential segments and cDNA clonicons were used as probe.
6、Made a cluster analysis of the polymorphic segments from AFLP-SDS-PAGE maps amplified by 5 prime pairs, and the 55 single chromosomes were classified to 10 groups. Then the 10 groups were hybridized with 134 clonicons from cDNA library, in order to proving the validity of the clusters, and a further Southern blot ensured the validity.
A preliminary classification was made and the homologous chromosome libraries were preliminarily constructed, but it is not enough in both integrity and quality, great afforts should be maken in case of construction of high-quality and integrity chromosome library.
The results in this study laid the foundation for the construction of accurate physic maps in future, and was very meaningful for subsequent genomic studys in Pomelo. And provided easier, faster and more accurate ways for genetic breeding and the location and cloning of useful genes in Pomelo.
引文
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