中文摘要

沉香是我国传统名贵香药，白木香（Aquilaria sinensis (Lour.) Spreng）是其主要的植物来源，是我国特有的珍稀濒危树种。由于天然沉香形成的概率低且过程缓慢，再加上人为的滥砍滥伐，其植物资源已濒临灭绝，无法满足人们对沉香的需求。近年来，香农在野外采香时发现了可以产生高品质奇楠沉香的白木香树，进行嫁接扩繁后，获得了可稳定遗传的奇楠种质。但由于奇楠种质来源混乱，且不同种质结香性能差异显著，严重制约了沉香产业的健康发展。因此，本研究从代表性奇楠种质结香性能考察、奇楠种质结香性能关联基因的筛选、鉴定结香性能分子标记的开发以及奇楠种质结香性能关联基因的生信分析四个方面，探究奇楠种质沉香生物合成的调控机制以及分子辅助育种方法，主要研究结果如下：

（1）奇楠种质间存在结香性能差距较大的优劣种质群体。选取QLX、TJ、YYZ等36种市面上主流的奇楠种质，利用钻孔法对36种奇楠种质进行人工结香，于6个月后收样。纵向剖开枝干，观察并测量奇楠种质的油脂扩散情况。从传统中药材质量评价方式出发，以油脂扩散长度为主、参考颜色等评价指标，综合评价36种奇楠种质的结香性能。结果表明，36种奇楠种质可以根据油脂扩散长度大于或小于2cm的标准划分为结香性能好与差两个种质群体。具体而言，QLX、TJ和JSY的颜色为黑褐色/棕褐色，结香趋势明显，且油脂将两孔间距离都连通，结香性能最好；ZTJ、XYW、YBW等16种种质的颜色为黑褐色/棕褐色，结香趋势明显，且油脂单向扩散距离大于2cm，结香效果相对较好；TX2、BHL、ZYZ等12种种质的颜色为黑褐色/棕褐色/棕色，结香趋势明显，且油脂单向扩散距离小于2cm，结香效果相对较差；XS3、HZZ、XS8、XS13和DWG的颜色为棕色/浅棕色，油脂未满溢出钻孔，沉香形成缓慢甚至停止，结香性能最差。

（2）筛选出影响结香性能差异的关键次生代谢物质合成调控基因。根据前期普通白木香和奇楠种质的比较转录组测序数据标准化差异基因后，构建加权共表达网络（Weighted Gene Co-Expression Network，WGCN），识别基因模块并关联基因模块与表型。KEGG功能富集分析表明，与倍半萜和色酮成分生物合成相关的差异表达基因（DEGs）的数量共计48个。根据RNA-seq数据的标准化值(log2(FPKM)) 绘制热图，筛选出16个奇楠种质次生代谢物质合成相关基因。qRT-PCR检测结果表明，CNFLAs、CNHMGR、CNPKS7、CNTPS15和CNTPS18这5个次生代谢物质合成DEGs在结香性能优良的种质中表达水平上调，与在其他种质的表达相比具有显著差异。

（3）开发出可以鉴定奇楠种质结香性能优劣的分子标记并初步建立基因型-表型的鉴定规则，形成奇楠种质分级鉴定技术。基于转录组数据中结香性能关键基因的全长序列和启动子序列，补充结香性能好和差的种质样品进行Sanger测序，以鉴定目标基因的序列多态性。选择有潜力的SNP位点进行KASP分型检测，得到可以区分种质结香性能优劣的分子标记，并通过鉴定不同来源的奇楠种质资源，验证分子标记的准确性。结果显示，排除双峰和无结果的片段，Sanger测序鉴定得到61个候选SNP位点；KASP分型开发检测出11个有效的分子标记；经过不同来源的奇楠种质实地结香验证，并总结实验结果形成奇楠种质结香性能鉴定规则，分子标记组合后建立的分级鉴定技术鉴定率为93.2%。

（4）探究影响结香性能差异的关键基因CNTPS18的结构与功能。根据白木香基因组信息，分段克隆并拼接得到影响结香性能的关键次生代谢物质合成基因CNTPS18全长1632bp的CDS序列。利用生物信息学分析软件，发现CNTPS18编码蛋白没有跨膜结构，也没有信号肽结构，属于不稳定性蛋白和亲水性蛋白；具有8个倍半萜合酶的保守结构域，预测位于奇楠种质的细胞质基质中；二级结构以α螺旋结构为主，三级结构与螺环二烯合成酶的一致性很高；亲缘关系上与同属的马来沉香Aquilaria malaccensis最为接近。

综上所述，本研究通过考察代表性奇楠种质的结香性能，划分不同种质结香性能优劣。在明确结香性能的基础上，根据转录组测序数据通过WGCNA分析和和qRT-PCR检测，筛选出影响结香性能差异的关键基因，利用Sanger测序和KASP分型技术开发出有效的分子标记并验证，最终形成奇楠种质分级鉴定技术。同时，通过全长序列的克隆和生信分析，进一步探究影响结香性能差异的次生代谢物质合成基因CNTPS18的结构和功能。这些结果为深入了解高品质奇楠沉香形成的分子机制提供了重要的基础，同时为奇楠种质育种技术的发展提供理论依据。

Abstract 

Aquilaria sinensis (Lour.) Spreng is a rare tree species unique to China, which is the only plant source of agarwood, known as the traditional precious Chinese medicine. Due to the inherently low probability and protracted process of natural agarwood formation, compounded by anthropogenic deforestation, the plant resources have been endangered , thereby failing to satisfy the people's demand for agarwood. In recent years, agarwood growers have discovered certain Aquilaria sinensis trees that can produce high-quality incense. And after grafting and expanding, the stable genetic germplasms have been obtained. However, the study on Chi-Nan germplasm breeding has been seriously constrained by the fact that the unclear source of its mother trees and the lower quality of  agarwood from the second-generation seedlings.Therefore, in this study, we investigated the regulation of agarwood biosynthesis and molecular-assisted breeding methods in the Chi-Nan germplasm from the examination of the agarwood formation properties of the representative Chi-Nan germplasm, the screening of the genes associated with agarwood formation properties, the development of molecular markers for the identification of agarwood formation properties, and bioblotting analysis of the genes associated with agarwood formation properties, and the main findings are as follows:

There are groups of superior and inferior germplasms with large disparities in agarwood formation properties among Chi-Nan germplasms. Nine representative Chi-Nan germplasms, including QLX, TJ, YYZ, and so on, were selected, and the samples were collected after 6 months of artificially wounded induction by using the drilling method. Branches and trunks were dissected to observe and measure the resin extension of the Chi-Nan germplasms, and photographs were taken. The agarwood formation properties of the nine qinan germplasm was comprehensively evaluated based on color, aroma, oil content and degree of resin extension. The results showed that QLX, TJ, YYZ, XGY and BHL were darker in color and higher in oil content; the extension distance of QLX and TJ was longer and the fragrance was more intense; and the pungency of QLX and TJ was found to be more pungent compared with the other germplasms after oral tasting. In conclusion, QLX and TJ had superior agarwood formation properties, followed by YYZ, XGY, and BHL; the rest of the germplasms had inferior ones.

It was screened that the secondary metabolites synthesized and regulated key genes were associated with the superiority or inferiority of agarwood formation properties. Comparative transcriptome sequencing of the ordinary Aquilaria sinensis and Chi-Nan germplasms was conducted, and KEGG functional enrichment analysis showed that the number of differentially expressed genes (DEGs) associated with the biosynthesis of sesquiterpene and chromone components totaled 48. Based on the normalized value (log2(FPKM)) of RNA-seq data, a heat map was drawn about these genes. We screened out 16 genes related to secondary metabolite synthesis in Chi-Nan germplasms from these genes. qRT-PCR verification results showed that the expression levels of five secondary metabolite synthesized DEGs, such as PKS7, TPS18, etc., were up-regulated in the germplasms of the superior agarwood formation properties, which was significantly different from that of the expression in other germplasms.

Completed the development and validation of molecular markers to differentiate between germplasm with superior or inferior agarwood formation properties. Based on the full-length sequences and promoter sequences of the genes associated with superior or inferior agarwood formation properties in the transcriptome data, Sanger sequencing was performed on additional samples of germplasm with superiority and inferiority to identify the sequence polymorphism of the target genes. Potential SNP loci were selected for kompetitive allele specific（KASP）typing to obtain molecular markers that could distinguish the superior and inferior agarwood formation properties of Chi-Nan germplasms, and the accuracy of the molecular markers was verified by identifying the Chi-Nan germplasms from different sources. The results showed that 61 candidate SNP loci were identified by Sanger sequencing after excluding double peaks and fruitless fragments; 11 effective molecular markers were detected by KASP typing; the identification rate was 93.2% after the combination of 11 molecular markers, as verified by the field conjugation of Chinaberry germplasm from different sources.

Investigation of the structure and function of CNTPS18, a key gene associated with the differences of agarwood formation properties. The full-length CDS sequence of CNTPS18, a secondary metabolite synthesis gene associated with agarwood formation properties, was cloned based on the transcriptome information, and analyzed bioinformatically to predict the amino acid sequence, promoter cis-elements, and protein structure and function.

In summary, this study investigated the key genes associated with the superiority and inferiority of agarwood formation properties and developed molecular markers to distinguish the agarwood formation properties of Chi-Nan germplasms. By examining the condition of agarwood formation in representative Chi-Nan germplasms, we were able to classify the agarwood formation properties of different Chi-Nan germplasms. Based on the transcriptome sequencing data and qRT-PCR verification, the key genes associated with the superiority and inferiority of agarwood formation properties were screened out, and effective molecular markers were developed and validated by using Sanger sequencing and KASP typing technology. Meanwhile, the structure and function of CNTPS18, a secondary metabolite synthesis gene associated with the agarwood formation properties, were further investigated by full-length sequence cloning and biosignature analysis. These results provide an important foundation for the in-depth understanding of the molecular mechanism of the formation of high-quality agarwood, and at the same time furnish a theoretical basis for the development of Chi-Nan germplasm breeding technology.