【作者】 王鹏飞；

【导师】 康国章；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2021， 博士

【摘要】 磷是植物生长发育所必需的大量营养元素之一,但土壤中有效磷含量不能满足作物正常生长发育的需求,磷缺乏已经成为影响作物产量和品质的一个重要问题。虽然施磷能大幅提高作物产量和品质,但由于大多数作物的磷肥利用效率较低,导致大部分磷肥被浪费,引起农业生产成本增加和环境污染。因此提高作物的磷肥利用效率,对于农业可持续发展与环境保护具有重要意义。普通小麦是世界分布最为广泛和重要的粮食作物,也是一个复杂的异源六倍体物种,但有关小麦的缺磷响应与分子调控网络却并未深入研究。本研究以普通小麦为研究对象,利用蛋白组学方法辨析出一个缺磷诱导表达的磷转运蛋白TaPHT1;9-4B,研究其在缺磷响应中的功能,并分离出调控其表达的上游转录因子,最后在小麦品种间鉴定出该基因的优异变异位点,开发其分子标记,为磷高效小麦育种提供了筛选标记。主要研究结果如下:1.TaPHT1;9-4B转运蛋白的辨析。小麦幼苗在缺磷胁迫条件下,地上部分的生长受到抑制,而根系生长则受到促进,与正常供磷的小麦幼苗相比,在磷缺乏第8天,小麦植株的生长指标(株高、根长、干重)和磷含量均出现显著差异。利用高通量i TRAQ蛋白组学技术比较了该时间点正常供磷与缺磷两处理小麦幼苗根和叶中的蛋白质组表达差异,共辨析出763个缺磷响应蛋白(根中363个,叶中400个)。这些蛋白主要参与了物质转运、信号转导、激素合成、物质代谢、胁迫与防御等生物学过程,表明小麦对磷缺乏响应是一个复杂的生理生化适应过程,并且根中和叶片内的差异表达蛋白种类具有较大差异,说明小麦器官之间对缺磷响应机制存在一定差异。根据最新公布的中国春小麦基因组数据库,对蛋白组学中鉴定到的磷缺乏响应蛋白进行染色组定位,有近四分之一(1015/4306,23.6%)的磷缺乏响应蛋白能够被准确地定位到染色体上,其中78.5%(797/1015)磷缺乏响应蛋白表现出部分同源拷贝之间表达不对称特性(单拷贝特异表达与多拷贝差异表达)。同时还发现4个磷转运蛋白(TaPHT1;3-5B、TaPHT1;6-5B、TaPHT1;9-4B和TaPT2)在缺磷小麦幼苗根系内显著上调表达,推测这些蛋白在小麦适应缺磷胁迫过程中起着重要作用,其中以TaPHT1;9-4B蛋白丰度上调最高。2.TaPHT1;9-4B转运蛋白的功能。TaPHT1;9-4B是一个包含12个跨膜结构域的高亲和磷转运蛋白,亚细胞定位在细胞膜上。编码该蛋白的基因三拷贝分别位于4A、4B和4D染色体上。它在一个磷酸转运功能缺失的酵母突变体MB192中表达,能够恢复突变体在低磷培养条件下的正常生长,其最适生长环境为弱酸性,表明TaPHT1;9-4B具有磷酸盐转运功能;通过大麦条纹花叶病毒诱导的基因沉默技术(BSMV-VIGS)使TaPHT1;9三拷贝(4A、4B、4D)在小麦体内表达受到抑制,发现沉默植株的磷吸收能力显著降低,小麦幼苗的生长受到抑制。同时TaPHT1;9-4B在水稻体内的异源表达能显著提高转基因植株的磷吸收能力,促进转基因植株在低磷与缺磷水培环境下的生长,还能促进转基因水稻对土壤中磷的吸收,提高转基因植株在低磷供给条件下的产量,提高磷肥利用效率。3.TaPHT1;9-4B基因上游转录因子的分离。以TaPHT1;9-4B启动子片段为诱饵,通过酵母单杂交(Y1H)对磷饥饿小麦幼苗根系c DNA文库进行筛选,获得了一个MYB转录因子。序列分析表明该转录因子编码基因位于7D染色体,与拟南芥中的At MYB4亲缘关系最近,命名为TaMYB4-7D。它是一个R2R3型的MYB家族转录因子,亚细胞定位于细胞核内,具有转录激活活性。通过Y1H与双荧光素酶报告基因检测试验(Dual-luciferase assay)证明它能够通过识别MYB结合位点(MBS)与TaPHT1;3-5B、TaPHT1;6-5B、TaPHT1;9-4B和TaPT2启动子结合并正向调控其表达。BSMV病毒诱导的TaMYB4基因沉默同样抑制了沉默植株的磷吸收,同时降低上述4个磷转运蛋白编码基因(TaPHT1;3-5B、TaPHT1;6-5B、TaPHT1;9-4B和TaPT2)的表达。4.TaPHT1;9-4B基因分子标记的开发。通过对62份六倍体小麦(AABBDD)、11份四倍体(AABB)和28份二倍体(BB或DD)小麦祖先种中的TaPHT1;9-4B和TaMYB4-7D基因及其直系同源基因的重测序,发现小麦从二倍体经过四倍体再到六倍体的进化过程中,MYB4-7D的基因序列(启动子与编码区)只在其二倍体祖先种中有少量变异,在六倍体中没有变异,说明MYB4-7D在进化过程中比较保守。PHT1;9-4B在进化过程中则经历了比较显著的演化与选择,其启动子与编码区在二倍体祖先种中存在较多变异(SNP与Indel),到四倍体再到六倍体中的变异则显著减少,并且启动子区域的变异显著多于编码区。在六倍体小麦中,TaPHT1;9-4B启动子(-1513bp～-1bp)中9个SNP能够紧密连锁形成四个单倍型(Hap1、Hap2、Hap3和Hap4),其中Hap3启动子具有最强的转录驱动能力。且Hap3单倍型小麦品种在低磷条件下具有更高的TaPHT1;9-4B转录水平与磷积累量,表明Hap3为磷吸收优异单倍型,进而开发出一个分子标记CAPS-799,它能够区分Hap3与其它单倍型,可作为磷高效小麦品种选育中的筛选标记。综上所述,本研究通过蛋白组学、转基因等分子生物学方法发现并证实了一个由磷转运蛋白(TaPHT1;3-5B、TaPHT1;6-5B、TaPHT1;9-4B、TaPT2)和TaMYB4-7D转录因子构成的分子模型,它能够参与小麦对缺磷胁迫的响应,为解析小麦的磷吸收和利用分子机制提供了一些新发现。同时,TaPHT1;9-4B优异等位基因的鉴定与CAPS分子标记的开发有助于磷高效小麦新品种的选育。更多还原

【Abstract】 Phosphorus is one of the most indispensible macronutrients for plant growth and development,because it is a major component of phospholipids and nucleic acids.Generally,Pi concentration in soil solution is frequently below the critical level needed by plants and Pi deficiency severely limits crop growth and yield.To improve crop yields,millions of tons of Pi fertilizers are applied annually to agricultural fields worldwide.However,only 10~20% of the applied Pi is effectively absorbed by plants due to the low utilization efficiency of Pi fertilizers in crops,while the remaining is lost,which not only increases agricultural costs but also causes environmental pollution.Therefore,improving the phosphate utilization efficiency of crops is greatly important to ensure the sustainable development of agriculture and environmental protection.Although bread wheat is the most widely cultivated and important staple food crop in the world,it is a complex polyploid model plant,and underlying molecular mechanism respone to Pi deficiency is still poorly understood.In this study,we applied proteomic approach to identify Pi deficiency-responsive protein species(PDPSs).Next,we identified and functionally characterized the Ta PHT1;9-4B transporter,and isolated its upstream transcription factors by using a series of molecular biological methods.Finally,we further identified the favored haplotype of Ta PHT1;9-4B in modern bread wheat cultivars and developed its functional molecular marker.The major results were summarized as follows:1.Identification of Ta PHT1;9 transporter.The wheat seedlings cultured in Pi-deficient conditions showed inhibited leaf growth but enhanced root length compared with the Pi-sufficient plants.Quantitative analysis of growth parameters(plant height,root length,shoot and leaf dry weight)and P concentrations further confirmed these changes.We applied the high throughput i TRAQ-based proteomic approach to survey the protein expression patterns of roots and leaf in the Pi-deficient and-sufficient wheatseedlings.A total of763 protein species(363 in root,400 in leaf)with significantly altered abundance(≥1.2-fold or ≤ 0.83-fold change)were identified and they were functionally involved in many biological processes(e.g.,transportation,metabolism,signal transduction,stress and defense,and function unknown).Moreover,there were significant differences between the PDPSs in roots and leaves,which indicated that the response mechanisms of Pi deficiency between the root and leaf organs were different to some extent.Genomic alignment of the identified protein species was studied using the genome assembly of Chinese Spring(CS).Among the4,013 identified protein species,1015 were assigned specifically to the unique homoeologs.A large proportion(78.5%,797/1015)of these protein species showed asymmetric expression pattern under Pi deficiency stress,including single homoeolog specific expression and multi homoeolog differential expression.Among the identified PDRPs,the abundance of four high affinity PHT1 proteins(Ta PHT1;3-5B,Ta PHT1;6-5B,Ta PHT1;9-4B,and Ta PT2)was significantly increasedin the roots of Pi-deficient wheat seedlings,with the average fold of induction being highest for Ta PHT1;9-4B,These results indicated that Ta PHT1;9-4B might play key role in response to Pi deficiency in wheat.2.Function of Ta PHT1;9 transporter.Ta PHT1;9-4B was a high-affinity Pi transporter characterized with 12 putative transmembrane(TM)domains and it localized at the plasma membrane.Three Ta PHT1;9 homoeologs were present on the 4A,4B,and 4D chromosomes of CS,respectively.In the MB192 yeast mutant strain defective in Pi absorption,the expression of Ta PHT1;9-4B restored its growth under low Pi conditions with the highest degree of complementation observed at p H 6.0,indicated that Ta PHT1;9-4B protein was characterized with Pi transport activity.The ectopic expression of Ta PHT1;9-4B in rice improved Pi uptake and transgenic plants growth under both Pi replete and deprived conditions,whereas the transient silencing of the Ta PHT1;9 gene in the wheat seedling by using barley stripe mosaic virus mediated gene silencing(BSMV-VIGS)decreased the Pi absorption and impaired wheat growth.In the pot experiment with four levels of P fertilizer,the grain yields and biomass of Ta PHT1;9-4B ectopic transgenic rice plants were also significantly higher than those of WT plants under P fertilizer-insufficient suppliment,and the P concentration in the root and straw of transgenic plants were also significantly higher than WT.These suggested that the Ta PHT1;9 was a candidate gene for the cultivation of phosphate use efficiency crops.3.Isolation of the upstream transcription factor for Ta PHT1;9 transporter.The promoter fragment of Ta PHT1;9-4B was used as bait to screen the c DNA library derived from Pi-deficient wheat roots by using yeast one hybrid(Y1H),and a R2R3-type MYB transcription factor Ta MYB4-7D was obtained.Its coding gene was located on 7D chromosome and the peotein phylogenetically related to At MYB4 transcription factor.Ta MYB4-7D transcription factor localized in the nucleus and possessed transcriptional activation activity.The Y1 H and dual-luciferase assay experiment furtherconfirmed that Ta MYB4-7D activated Ta PHT1;3-5B,Ta PHT1;6-5B,Ta PHT1;9-4B and Ta PT2 by directly binding to MBS elements in their promoter.Silencing Ta MYB4 by BSMV-VIGS conferred more sensitivity to low Pi or Pi deficiency and downregulated the transcript levels of the Ta PHT1;3,Ta PHT1;6,Ta PHT1;9 and Ta PT2 genes.4.Development of the Ta PHT1;9 molecular marker.The promoter and coding region of Ta PHT1;9-4B,Ta MYB4-7D and their orthologs were resequence in the 62 common wheat cultivars,11 tetraploid,and 28 diploid wheat ancestral relatives.Sequence analysis showed that there were only several single nucleotide polymorphisms(SNPs)in the promoterand coding region of MYB4-7D in diploid species,and no polymorphism was found in hexaploid wheat,implying highly conservation of the MYB4-7D in the evolutionary process.However,a large number of polymorphic sites(SNP and indel)were detected in the promoter and coding region of Ta PHT1;9-4B and its ortholog genes.Systematic evolutionary analysis suggested that genetic diversity of PHT1;9-4B decreased significantly during the evolution from diploid to polyploid species,and genetic diversity of promoter region was significantly higher than coding region.Four promoter haplotypes were identified for Ta PHT1;9-4B in modern wheat cultivars.Hap3,the favored haplotype,showed significant positive associations with Ta PHT1;9-4B transcript level and phosphorus content in wheat plants,and Hap3 promoter had stronger activity.A functional marker CAPS-799 was developed to discriminate Hap3.In summary,this study uncovered a molecular module composed of Ta MYB4-7D transcription factor and four PHT1,which required for maintaining efficient Pi acquisition and plant growth under Pi limiting conditions.Our work sheds new light on the molecular mechanism controlling Pi acquisition and utilization in bread wheat.In addition,the favored haplotype of Ta PHT1;9-4B promoter and the developed CAPS-785 markermay facilitate the development of P efficient wheat cultivars in the future.更多还原