【作者】 胡薇；

【导师】 林思祖；

【作者基本信息】 福建农林大学 ， 森林培育， 2010， 博士

【摘要】 黑木相思(Acacia Melanoxylon)属含羞草科(Mimosaceae)金合欢属(Acacia Mill.)。自然分布于澳大利亚布里斯班以南,至南澳州的东南部和塔斯马尼亚岛,是近年来我国南方引种的主要用材树种之一,它具有适应性强、速生丰产、用途广泛等优点。但由于种源地属热带地区,大多种源对低温仍较为敏感,引种实践证明,在闽北及其以北地区长势较差,闽北低温是其种苗越冬的主要限制因子;另外,其实生苗在栽培中性状分化十分严重,制约了高产质优的黑木相思人工林的发展。因此,研究黑木相思优树遗传多样性、抗寒性,鉴定寒胁迫诱导下差异表达基因的生物学功能,可为进一步阐明黑木相思的抗寒机理,培育出性状稳定的抗寒相思优良品系提供理论依据;这对黑木相思优良种源引种适宜区的选择、优异抗逆林木的早期选择和种质资源库建设具有重要而深远的意义。本研究采用ISSR分子标记技术分析了黑木相思优树遗传多样性,采用抗寒评估技术对供试黑木相思优树进行了抗寒性评估,采用cDNA-AFLP技术对黑木相思寒胁迫诱导差异表达基因进行了研究,主要结果如下:1建立了黑木相思基因组DNA快速提取方法采用1.5×CTAB法提取黑木相思叶状柄的基因组DNA,其分子大小约为48Kb,得率约为180-697.5μg﹒g-1, A260/A280=1.75～1.955,无需经RNase处理,可直接用于限制性酶切和ISSR分析。2采用均匀设计优化和建立了黑木相思ISSR-PCR体系,筛选了多态性高、稳定性好的ISSR引物10条,并对其扩增程序和引物退火温度进筛选优化。(1)黑木相思ISSR-PCR的体系:在20μL反应体系中,含引物0.30μmol·L-1、Mg2+ 3.00mmol·L-1,dNTP 0.15mmol·L-1、Taq DNA聚合酶0.75U和模板DNA2.00ng·μL-1。(2)黑木相思ISSR-PCR的扩增程序:94℃预变性5min;接着进行33个循环:94℃变性35s,52-61.5℃退火52s,72℃延伸90s;循环结束后,72℃延伸10min。3黑木相思优树遗传多样性ISSR分析的验证用ISSR标记技术,采用上述优化的黑木相思ISSR-PCR反应体系和循环参数,从加拿大哥伦比亚大学(UBC)公布的第9套(共计100条ISSR引物)中筛选出的10条引物,对37个黑木相思优树进行遗传多样性分析,结果显示: 37个黑木相思优树之间存在较高的遗传多样性;而引自福建不同栽培地域的优树之间的遗传多样性则相对较小,这与引种黑木相思种源背景资料相一致,与引种栽培事实相符。37个黑木相思优树间的Nei & Li(1979)遗传距离表明:供试的37个黑木相思优树之间存在较大的遗传分化,这种遗传分化显现出显著的地域性;相对而言,3个来自建瓯的抗寒优树相对具有较高的遗传多样性;16个引自广东的优良无性系根孽苗优树具有较高的遗传多样性和较小的遗传分化;7个引自广东的优良无性系采种实生苗优树遗传多样性最低,且遗传分化较大;这与引种黑木相思种源背景和种源试验的栽培客观事实相符。研究结果表明:ISSR分子标记技术适用于黑木相思的遗传多样性分析。基于ISSR标记的黑木相思37个优树的聚类分析表明: 3个引自建瓯的抗寒优树与其它34个黑木相思优树亲缘关系可能较远;引自广东的16个优良无性系根孽苗优树与其半同胞优树(7个优良无性系采种实生苗优树)优树聚为一支,说明了它们之间的亲缘关系;这与种源背景和栽培事实相符。研究结果表明:ISSR分子标记技术适用于黑木相思的亲缘关系鉴定。另外,筛选出的10个引物对3个抗寒优树的扩增,共扩增出了48个特异遗传位点,其中3个抗寒优树共有的特异遗传位点有22个,可作为区别于其他34个优树的ISSR分子标记,另外26个特异遗传位点可与22个共有特异遗传位点配合使用,作为3个抗寒优树之间相互区分的ISSR标记。4黑木相思的抗寒性评估应用电导法配以Logistic方程求拐点温度作为黑木相思的低温半致死温度,能较直观且准确地反映黑木相思的抗寒力和所能忍耐的低温极限,可作为黑木相思抗寒性评估的有效方法。黑木相思的抗寒性大小与其寒胁迫过程中,细胞中MDA含量累积幅度相关,即抗寒性强的无性系,其细胞中MDA含量累积幅度小,抗寒性弱的无性系,其细胞中MDA含量累积幅度大,因此,寒胁迫中,黑木相思细胞中MDA含量累积幅度大小可作为其抗寒性评估的检测指标。低温胁迫下黑木相思可溶性糖含量变化与其抗寒性的关系复杂,部分种源细胞可溶性糖累积与其抗寒性呈负相关;低温下黑木相思细胞可溶性糖的累积作用,对提高其抗寒性可能是间接的,更多的可能趋向于诱导启动抗寒的其它过程,间接提高抗寒性。低温胁迫下黑木相思脯氨酸含量变化与其抗寒性的关系复杂,部分种源细胞脯氨酸累积与其抗寒性呈负相关;低温下黑木相思细胞脯氨酸的累积作用,更多的可能趋向于提高细胞的渗透浓度,进而诱导启动抗寒的其它过程,或只是低温胁迫的结果。5建立了适于cDNA-AFLP的黑木相思组培苗总RNA快速提取方法采用改良CTAB-异硫氰酸胍裂解法能有效地控制多酚、多糖、蛋白质以及DNA等对所提取总RNA的污染,所得总RNA质量高、完整性好;紫外分光光度检测,A260/A280比值为1.96-2.0,A260/A230比值1.98-2.2,总RNA得率为118.4-213.6μg.g-1,电泳检测,28SrRNA亮度约为18SrRNA的两倍,所提RNA质量可以满足dscDNA合成和cDNA-AFLP等后续分子操作要求。6建立了黑木相思双低频酶cDNA-AFLP反应体系以黑木相思组培苗为实验材料,对影响cDNA-AFLP反应体系的关键因素进行了研究,建立了适用于黑木相思的cDNA-AFLP分析体系。结果表明:适用于黑木相思dscDNA的限制性酶切组合为EcoRI和PstⅠ,dscDNA酶切用量为500 ng。用作预扩增模板的连接产物稀释倍数为1倍,用作选择性扩增模板的预扩增产物稀释倍数为50倍。7寒胁迫诱导黑木相思差异表达基因的分离和鉴定采用cDNA-AFLP技术,得到黑木相思响应低温胁迫差异表达转录谱,并对图谱中有差异变化的147个TDF测序,测序结果在NCBI上进行同源性比对分析,其中下调表达基因为7个,上调表达基因为140个,这些基因主要有:锌指蛋白、GTP酶激活蛋白、鸟嘌呤核苷酸交换因子、蛋白磷酸酶-2A、MYB结构域蛋白、转录因子NAC、类受体蛋白激酶、蛋白质酪氨酸磷酸酶-受体型B、核糖核苷二磷酸还原酶、细胞色素P450、羧酸酯水解酶、核酮糖-1,5-二磷酸羧化酶、尿素酶辅助蛋白ureG、质膜H+-ATPase蛋白、核糖体蛋白S7、细胞周期蛋白、硫氧还蛋白等。本研究结果为进一步研究黑木相思的抗寒机理及差异表达TDFs相应基因在黑木相思应答低温中可能的作用研究奠定了基础。更多还原

【Abstract】 Acacia Melanoxylon is a Mimosaceae Acacia Mill, naturally distributed from south Brisbane, Australia to the south-east of South Australia and Tasmania. It is one of the main timber tree species in South China, which was imported within recent years. It features easy adaptation, high yield and broad range of usages and so forth. However, because of original from tropical regions, the most of them are still very sensitive to low temperature. Real practice has proved that Acacia Melanoxylon imported into the north Fujian & the further northern regions grows poorly. Low temperature is the main limit in wintertime. In addition, it’s seedling traits in cultivation with significant differentiation, restricting plantation development of Acacia Melanoxylon of the high-yield and high-quality. Therefore, the study of Acacia melanoxylon plus tree, genetic diversity, cold tolerance and identification of biological function of genes expressed differentially under cold stress can provide a theoretical basis for further clarification of the cold-resistance mechanism of Acacia melanoxylon and cultivating good character and stability of the cold Acacia strains. In this study, ISSR molecular marker has been used to analysis of genetic diversity in Acacia melanoxylon plus tree, cold-assessment techniques have been used to test cold tolerance of Acacia melanoxylon, cDNA-AFLP technology has been used to to analysis differentially expressed genes in cold-induced .The main results are as followings:1 A fast and convenient method for extraction of DNA from Acacia Melanoxylon is established.1.5×CTAB method is considered as one of fast and convenient methods for extraction of DNA in young fresh leaves of Acacia Melanoxylon . The results indicates that 180-697.5μg·g-1DNA are obtained from a phyllode , their molecular weights are about 48 Kb, and the A260/A280 of DNA are 1.75～1.955,the DNA is suitable for digesting with restriction enzyme and ISSR reaction without purifying by RNase.2 ISSR-PCR system of Acacia Melanoxylon has been established and optimized with uniform design, screening ten of ISSR primers with rich polymorphism and good stability, optimizing ISSR amplification procedures and primer annealing temperature of Acacia Melanoxylon.(1) ISSR-PCR system of Acacia Melanoxylon: Each 20μL ISSR-PCR amplification reaction solution is consisted of 3.00 mmol·L-1 Mg2+ , 0.15 mmol·L-1 dNTP, 0.30μmol·L-1 Primer , 0.75U Taq DNA polymerase , 2.00 ng·μL-1DNA template.(2) ISSR amplification procedures of Acacia Melanoxylon: The suitable ISSR-PCR procedure was 1 cycle of pre-denaturing for 5 min at 94℃, 33 cycles of denaturing for 35s at 94℃, annealing for 52s at 52-61.5℃, extending for 90s at 72℃and the last cycle of extending for 10 min at 72℃.3 Verification to Genetic diversity of Acacia Melanoxylon based on ISSR markersGenetic diversity among 37 Acacia Melanoxylon plus trees is detected by ISSR markers. The results reveal a high level of genetic diversity among 37 Acacia Melanoxylon plus trees, having a low level of genetic diversity between different Acacia Melanoxylon plus trees that are planted in different region in Fujian. This results are matching background information of Acacia melanoxylon and cultivation facts. The Nei & Li(1979) genetic distance among 37 Acacia Melanoxylon plus trees reveals a high genetic differentiation among 37 Acacia Melanoxylon plus trees. Relatively, 3 plus trees from Jianou, Fujian have a high level of genetic diversity; 16 plus trees of good clones from Guangdong have a high level of genetic diversity with a low genetic differentiation. 7 seedlings of good clones from Guangdong have the lowest level of genetic diversity. The results show that: ISSR molecular marker technique is suitable to be applied to analysis of the genetic diversity of Acacia Melanoxylon .Cluster Analysis show that: genetic relationship between 3 plus trees from Jianou, Fujian and the other 34 Acacia Melanoxylon plus trees maybe is far. 16 plus trees of good clones from Guangdong cluster together with half-sib 7 seedlings of good clones, which reveals that they are genetically related. This is matching background information of Acacia melanoxylon and cultivation facts. Study shows that: ISSR molecular marker technique is suitable to be used for identification of genetic relationship of Acacia Melanoxylon .In addition, 10 primers for amplification of three cold-resistance plus tree are amplified in 48 specific genetic loci, in which a total of three cold-resistance plus tree-specific genetic loci, 22, can be used as difference from other 34 plus tree of ISSR molecular markers; Another 26-specific genetic loci with a total of 22 specific genetic loci used in conjunction, can be used as ISSR molecular markers to distinguish between three cold-plus tree.4 Cold-resistance assessments on Acacia melanoxylonThe relative electrical conductivity of Acacia Melanoxylon under low- temperature treatment was measured by electrical conductivity method. The turning point temperature (semi lethal temperature) measured by Logistic curve is used as the index of cold-tolerance, may directly and effectively show cold resistance and low temperature limit of Acacia melanoxylon, so this method is an effective one for cold-resistance assessment on Acacia melanoxylon.Cold resistance of Acacia melanoxylon is associated with the cumulative rate of MDA content of cells under low-temperature treatment. MDA content of cells accumulated a small extent in cold tolerant clones. Meanwhile, MDA content of cells is accumulated a large extent in weak cold resistance clones. Results show that cumulative rate of MDA content of cells can be used as detective index of cold-resistance assessment on Acacia melanoxylon.The relationship between soluble sugar content and cold resistance is complicated. Some provenances of Acacia melanoxylon, soluble sugar content are negatively correlated with cold resistance, revealing indirect effect of soluble sugar accumulation in the cold resistance .The relationship between proline content and cold resistance is complicated. Some provenances of Acacia melanoxylon, proline content are negatively correlated with cold resistance. Increasing of proline content leads to increase osmotic, inducing other processes of cold resistance or result low-temperature stress only.5 A fast method suited to extract total RNA applied to cDNA-AFLP from plantlets of Acacia melanoxylon cultured in vitroThe modified CTAB- guanidinium isothiocyanate method can efficiently remove polyphenol, polysaccharide, proteins and DNA that often contaminate with RNA. The high-quality and integrity total RNA acquired the 28S rRNA, which is approximately two times as light as the 18S rRNA with 1.96-2.0 of OD260/OD280 ratio and 1.98-2.2 of OD260/OD230 ratio, and the total RNA yield of 118.4-213.6μg.g-1, and the quality of extracted RNA meet the requirement of subsequent molecular biology reactions as in dscDNA synthesis and cDNA-AFLP6 Establishment of cDNA-AFLP analysis system with two low- frequency cut Enzyme in Acacia MelanoxylonAn optimized system is established after studying on the factors affecting cDNA-AFLP analysis system of Acacia Melanoxylon cultured in vitro. The results indicates that the restriction enzyme combination is EcoR I and PstⅠ,the dosage of dscDNA is 500 ng ,the ligation products used as template of pre-amplification are diluted to one time ,and the pre-amplification products used as template of select-amplification are diluted to50 times. 7 Isolation and identification of differentially expressed genes cold -induced in Acacia melanoxylonThe transcription profile of Acacia Melanoxylon response to cold stress is analyzed by cDNA-AFLP, and 147 differentially expressed TDFs (transcript-derived fragments) is sequenced functional annotated by Blast in NCBI. And the TDFs are further classified into 2 subgroups:Ⅰup-regulated expression (140)Ⅱdown-regulated expression (7). There are zinc finger protein, GTPase activating protein, guanine nucleotide exchange factor, protein phsophatase-2A, myb domain-containing protein, transcription factor NAC, receptor-like protein kinase (RL Ks), protein tyrosine phosphatase,receptor B(PTPRB), Ribonucleoside-diphosphate reductase, ribulose-1,5-bisphosphate carboxylase, cytochrome P450, carboxylic ester hydrolase, urease accessory protein ureG, H+-ATPase, ribosomal protein S7, Cyclin-related protein, Thioredoxin(Trx).The results of this study will build up the foundation for study on the mechanism of cold resistance and the possible role of responsing to low-temperature of TDFs corresponding to differentially expressed genes in Acacia melanoxylon.更多还原