小白菜耐盐性的遗传分析与耐盐相关基因的差异表达研究
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摘要
蔬菜耐盐品种的选育和利用将在提高分布在耕地中的盐碱地的利用、增加单位面积产量和提高生产效益的同时，改善农田生态环境，丰富盐碱地分布地区的蔬菜多样性。本论文以小白菜为研究对象，通过对其耐盐性遗传规律和耐盐相关基因表达的研究，认识其耐盐机理，挖掘耐盐相关基因，以期为通过杂交育种或分子育种进行小白菜等蔬菜作物的耐盐性改良提供理论依据。
本研究以8个具有不同耐盐水平的小白菜自交系为试验材料，利用完全双列杂交Ⅱ配制杂交组合，采用GriffingⅡ及Hayman法对小白菜耐盐性的配合力和基因效应进行了初步分析。结果表明，小白菜耐盐性的一般配合力和特殊配合力都达到了差异极显著水平。通过对小白菜不同品种耐盐性的一般配合力和特殊配合力的效应值估算及其比较，发现两个一般配合力高的亲本相配获得的F_1的性状值可能高，另外，两个一般配合力都不高的亲本，相配后的特殊配合力和F_1的性状值亦可能很高。小白菜耐盐性遗传符合“加性—显性”遗传模型。遗传效应中存在加性效应和显性效应，但以显性效应为主，且可能存在超显性。小白菜耐盐性主要表现为显性，敏盐性为隐性，耐盐性可能由其它基因调控下的寡基因控制。因此，在小白菜耐盐性育种中，一方面要选择一般配合力高的亲本，另一方面亦要注意选择特殊配合力高的组合，以利通过杂交优势育种育成耐盐一代杂种。
以2个小白菜耐盐和中耐品种为试材，采用mRNA差异显示技术，通过2对锚定引物和随机引物组合对不同盐浓度诱导和盐诱导不同时间耐盐相关基因差异表达进行研究。通过目测比较相关差异带的消长和深浅，发现不同浓度NaCl诱导下差异片段表达量的大小顺序为1.6％＞1.2％＞2.0％。1.6％NaCl诱导6h时差异表达片段表达量较大：12h表达量略有下降，但之后，随着诱导时间的增加，表达量又开始逐步增加：到48h时表达量趋于稳定；72h表达量又有所下降。结合耐盐表型鉴定结果，认为1.6％的NaCl诱导48h比较适合小白菜耐盐相关基因的差异表达分析。这些研究结果为我们认识耐盐相关基因的表达程序和特点，制定有效的耐盐性研究策略提供了理论基础。
对4个耐盐水平不同的自交系及其F_1代的四种杂交类型，即耐×耐、不耐×中耐、耐×不耐、耐×中耐，分别选取1.6％NaCl盐诱导48 h和非诱导对照的植株的相同部位的嫩叶进行mRNA差异显示分析。15对引物组合共产生2512条带，其中差异带共116条。发现亲本与F_1代杂交种之间在盐诱导相关基因的差异表达上存在明显的区别。而且，这种区别与杂种一代耐盐性的表现有着密切关系。这有助于我们理解植物耐盐杂种优势的理论基础，以指导杂种优势育种。
以2个小白菜耐盐和中耐品种为试材，取1.6％NaCl诱导48h与非诱导对照的植株的相同部位的嫩叶进行mRNA差异显示分析，78对引物组合在两品种中产生了10种表达类型及101条与盐诱导相关的差异带。挑选经反复PCR反应及Northern杂交鉴定为阳性的13个表达量高、易于回收的盐诱导差异表达片段进行克隆和测序，得到了7个差异片段的序列。BLAST同源序列比对发现：cDNA片段38和68分别与油菜抗冻和萝卜抗旱的表达基因有90％和95％的同源性，显示cDNA片段38、68与植物中逆境胁迫下的抗性表达基因有关。cDNA片段03与cDNA片段27的同源性高，且分别与拟南芥中钙依赖型蛋白激酶的mRNA有85％和86％的同源性；cDNA片段73与拟南芥蛋白激酶相关mRNA有90％同源性；cDNA片段79与拟南芥中参与防御反应负调控的保守MAPKK激酶有90％同源性和促分裂原活化蛋白激酶的激酶之激酶(MAPKKK)mRNA有90％同源性：推测cDNA片段03、27、73、79可能与植物受到外界环境胁迫时参与信号传导途径的表达基因有关。cDNA片段85可能是植物中新发现的基因，它与Homo sapiens基因组DNA中肝细胞、结肠直肠细胞和非小细胞肺癌的抗癌基因99％同源。耐盐品种盐诱导相关cDNA片段及序列的获得为我们挖掘和利用耐盐相关基因及深入认识植物的耐盐机理奠定了一定的基础。
Development and use of the vegetable varieties with salinity tolerance will increase the utilization of the saline soil distributing in arable land and the productivity of vegetables, improve the entironment on farmland, enrich the vegetable diversity in saline area. Studies on the genetics and gene expression of salinity tolerance in Pak-choi [.Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee.] under salt stress were carried out in order to understand the mechanism of salinity tolerance in plant, mine the related genes of salinity tolerance, and use heterosis or molecular breeding to enhance the salinity tolerance in vegetables.
Genetics of salinity tolerance in Pak-choi was investigated by a eight-parent complete diallel cross II design only including direct crosses. Combining ablity and genetic component analysis were estimated by using Griffingll and Hayman methods. Estimate of combining ability for salinity tolerance showed there were highly significant differences in GCA and SCA. Estimates of GCA and SCA effects of different varieties or combinations showed that not noly the parents with high GCA could produce good F! with high SCA, but also the parents with poor GCA could produce good combinations with high SCA. Inheritance of salinity tolerance in Pak-choi fitted to the "additive-dominant" model. There were additive effect and dominant effect in genetic effects. However, the dominant effect played a major role and overdominance may exist in salinity tolerance exhibition. Salinity tolerance was mainly controlled by dominant genes and salinity sensitively was controlled by recessive genes. Inheritances of salinity tolerance may be controlled by few major genes under the regulation of other genes. Selection should be focused on both the parents with high GCA and the higher SCA combinations in heterosis breeding on salinity tolerance improvement in Pak-choi.
The salinity tolerance related genes expression under salt stress of certain intervals and different salt levels in two varieties was studied with two primer combinations by DDRT-PCR. Expression intensity under 1.6% NaCl were stronger than that under 1.2% and 2.0% NaCl. Expression reached the highest level for 6h salt stress. Expression level was less at 12h than 6h. But expression increased with the time under stress from 12h. It tended to be steady at 48h. It decreased after 72h. It was suitable to study the differential expression of the salinity tolerance related genes in Pak-choi for 48h under 1.6%NaCl stress. These results above would help us know the procedure and characteristics of the expression of the salinity tolerance related genes, and make effective stratege for salinity tolerance study in Pak-choi.
The mRNA differential display was used to show the differential expression among the two salt-tolerant, one moderate salt-tolerant and one salt-sensitive parents and their four kinds of F1: tolerant tolerant, tolerant x moderate tolerant, sensitive x sensitive and tolerant x sensitive under 1.6%NaCl stress and non-salt stress. Fifteen primer combinations generated 116 differential cDNA fragments in 2512 cDNA fragments in total. Significant differences of gene expression among the parents and Ft were observed under salt stress. These differences were related to the phenomena of salinity tolerance. The results helped us understand the heterosis theoretically and guide the heterosis breeding.
The mRNA differential display was used to screen the related transcriptic drived fragments of salt tolerance in salt-tolerant and moderate salt-tolerant varieties under the 1.6% salt stress and non-salt stress. Seven-eight primer combinations generated 101 differential cDNA fragments which were divided into 10 expression types. After they were proved to be positive by repeated PCR and northern blotting, thirteen cDNA fragments were reamplified by PCR, cloned and sequenced. Seven cDNA sequences were gained. BLAST analysis from GenBank and Brassica datebase found cDNA fragment 38 and 68 were 90% and 95% homologous to the expression genes i
本研究以8个具有不同耐盐水平的小白菜自交系为试验材料，利用完全双列杂交Ⅱ配制杂交组合，采用GriffingⅡ及Hayman法对小白菜耐盐性的配合力和基因效应进行了初步分析。结果表明，小白菜耐盐性的一般配合力和特殊配合力都达到了差异极显著水平。通过对小白菜不同品种耐盐性的一般配合力和特殊配合力的效应值估算及其比较，发现两个一般配合力高的亲本相配获得的F_1的性状值可能高，另外，两个一般配合力都不高的亲本，相配后的特殊配合力和F_1的性状值亦可能很高。小白菜耐盐性遗传符合“加性—显性”遗传模型。遗传效应中存在加性效应和显性效应，但以显性效应为主，且可能存在超显性。小白菜耐盐性主要表现为显性，敏盐性为隐性，耐盐性可能由其它基因调控下的寡基因控制。因此，在小白菜耐盐性育种中，一方面要选择一般配合力高的亲本，另一方面亦要注意选择特殊配合力高的组合，以利通过杂交优势育种育成耐盐一代杂种。
以2个小白菜耐盐和中耐品种为试材，采用mRNA差异显示技术，通过2对锚定引物和随机引物组合对不同盐浓度诱导和盐诱导不同时间耐盐相关基因差异表达进行研究。通过目测比较相关差异带的消长和深浅，发现不同浓度NaCl诱导下差异片段表达量的大小顺序为1.6％＞1.2％＞2.0％。1.6％NaCl诱导6h时差异表达片段表达量较大：12h表达量略有下降，但之后，随着诱导时间的增加，表达量又开始逐步增加：到48h时表达量趋于稳定；72h表达量又有所下降。结合耐盐表型鉴定结果，认为1.6％的NaCl诱导48h比较适合小白菜耐盐相关基因的差异表达分析。这些研究结果为我们认识耐盐相关基因的表达程序和特点，制定有效的耐盐性研究策略提供了理论基础。
对4个耐盐水平不同的自交系及其F_1代的四种杂交类型，即耐×耐、不耐×中耐、耐×不耐、耐×中耐，分别选取1.6％NaCl盐诱导48 h和非诱导对照的植株的相同部位的嫩叶进行mRNA差异显示分析。15对引物组合共产生2512条带，其中差异带共116条。发现亲本与F_1代杂交种之间在盐诱导相关基因的差异表达上存在明显的区别。而且，这种区别与杂种一代耐盐性的表现有着密切关系。这有助于我们理解植物耐盐杂种优势的理论基础，以指导杂种优势育种。
以2个小白菜耐盐和中耐品种为试材，取1.6％NaCl诱导48h与非诱导对照的植株的相同部位的嫩叶进行mRNA差异显示分析，78对引物组合在两品种中产生了10种表达类型及101条与盐诱导相关的差异带。挑选经反复PCR反应及Northern杂交鉴定为阳性的13个表达量高、易于回收的盐诱导差异表达片段进行克隆和测序，得到了7个差异片段的序列。BLAST同源序列比对发现：cDNA片段38和68分别与油菜抗冻和萝卜抗旱的表达基因有90％和95％的同源性，显示cDNA片段38、68与植物中逆境胁迫下的抗性表达基因有关。cDNA片段03与cDNA片段27的同源性高，且分别与拟南芥中钙依赖型蛋白激酶的mRNA有85％和86％的同源性；cDNA片段73与拟南芥蛋白激酶相关mRNA有90％同源性；cDNA片段79与拟南芥中参与防御反应负调控的保守MAPKK激酶有90％同源性和促分裂原活化蛋白激酶的激酶之激酶(MAPKKK)mRNA有90％同源性：推测cDNA片段03、27、73、79可能与植物受到外界环境胁迫时参与信号传导途径的表达基因有关。cDNA片段85可能是植物中新发现的基因，它与Homo sapiens基因组DNA中肝细胞、结肠直肠细胞和非小细胞肺癌的抗癌基因99％同源。耐盐品种盐诱导相关cDNA片段及序列的获得为我们挖掘和利用耐盐相关基因及深入认识植物的耐盐机理奠定了一定的基础。
Development and use of the vegetable varieties with salinity tolerance will increase the utilization of the saline soil distributing in arable land and the productivity of vegetables, improve the entironment on farmland, enrich the vegetable diversity in saline area. Studies on the genetics and gene expression of salinity tolerance in Pak-choi [.Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee.] under salt stress were carried out in order to understand the mechanism of salinity tolerance in plant, mine the related genes of salinity tolerance, and use heterosis or molecular breeding to enhance the salinity tolerance in vegetables.
Genetics of salinity tolerance in Pak-choi was investigated by a eight-parent complete diallel cross II design only including direct crosses. Combining ablity and genetic component analysis were estimated by using Griffingll and Hayman methods. Estimate of combining ability for salinity tolerance showed there were highly significant differences in GCA and SCA. Estimates of GCA and SCA effects of different varieties or combinations showed that not noly the parents with high GCA could produce good F! with high SCA, but also the parents with poor GCA could produce good combinations with high SCA. Inheritance of salinity tolerance in Pak-choi fitted to the "additive-dominant" model. There were additive effect and dominant effect in genetic effects. However, the dominant effect played a major role and overdominance may exist in salinity tolerance exhibition. Salinity tolerance was mainly controlled by dominant genes and salinity sensitively was controlled by recessive genes. Inheritances of salinity tolerance may be controlled by few major genes under the regulation of other genes. Selection should be focused on both the parents with high GCA and the higher SCA combinations in heterosis breeding on salinity tolerance improvement in Pak-choi.
The salinity tolerance related genes expression under salt stress of certain intervals and different salt levels in two varieties was studied with two primer combinations by DDRT-PCR. Expression intensity under 1.6% NaCl were stronger than that under 1.2% and 2.0% NaCl. Expression reached the highest level for 6h salt stress. Expression level was less at 12h than 6h. But expression increased with the time under stress from 12h. It tended to be steady at 48h. It decreased after 72h. It was suitable to study the differential expression of the salinity tolerance related genes in Pak-choi for 48h under 1.6%NaCl stress. These results above would help us know the procedure and characteristics of the expression of the salinity tolerance related genes, and make effective stratege for salinity tolerance study in Pak-choi.
The mRNA differential display was used to show the differential expression among the two salt-tolerant, one moderate salt-tolerant and one salt-sensitive parents and their four kinds of F1: tolerant tolerant, tolerant x moderate tolerant, sensitive x sensitive and tolerant x sensitive under 1.6%NaCl stress and non-salt stress. Fifteen primer combinations generated 116 differential cDNA fragments in 2512 cDNA fragments in total. Significant differences of gene expression among the parents and Ft were observed under salt stress. These differences were related to the phenomena of salinity tolerance. The results helped us understand the heterosis theoretically and guide the heterosis breeding.
The mRNA differential display was used to screen the related transcriptic drived fragments of salt tolerance in salt-tolerant and moderate salt-tolerant varieties under the 1.6% salt stress and non-salt stress. Seven-eight primer combinations generated 101 differential cDNA fragments which were divided into 10 expression types. After they were proved to be positive by repeated PCR and northern blotting, thirteen cDNA fragments were reamplified by PCR, cloned and sequenced. Seven cDNA sequences were gained. BLAST analysis from GenBank and Brassica datebase found cDNA fragment 38 and 68 were 90% and 95% homologous to the expression genes i
引文
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