Somatic embryogenesis (SE) is an important factor influencing the in vitro regeneration efficiency in litchi (Litchi chinensis Sonn.). Although putrescine (Put) and D-arginine (D-Arg) supplementation can regulate embryonic callus (EC) proliferation and somatic embryo induction in litchi, the underlying mechanism remains unclear. This study aimed to investigate the effects of exogenous Put and D-Arg on the histomorphological changes, polyamine (PA) contents, and PA metabolism-associated enzyme activities of 'Feizixiao' litchi EC. ECs were cultured on original proliferation medium (M3) and M3 supplemented with 15.0 mg·L−1 Put (P3) or 0.35 g·L−1 D-Arg (Ar3) for three weeks. Exogenous Put and D-Arg significantly increased the EC proliferation rate. Put reduced the numbers of induced somatic embryos and regenerated plantlets, while D-Arg had the opposite effects. Histomorphological analysis showed that exogenous Put or D-Arg inhibited proembryo differentiation during EC proliferation. On the M3 medium, EC cells first divided into two cells and then sequentially differentiated through the multicellular proembryo, globular, heart-shaped, and cotyledonary embryo stages. On P3 and Ar3 media, embryonic cells enlarge more significantly, underwent repeated cell divisions, and subsequently formed a meristematic mass from which dicotyledonous embryos initiated. P3 and Ar3 media both enhanced the endogenous PA contents and decreased the endogenous ornithine decarboxylase (ODC) and polyamine oxidase (PAO) activities. Exogenous Put inhibited the endogenous arginine decarboxylase (ADC) activity and enhanced the diamine oxidase (DAO) activity, while exogenous D-Arg had the opposite impact. In further experiments, ECs from P3 and Ar3 medium were subsequently cultured on M3, P3 and Ar3 media, respectively. In the control, ECs from M3 medium were cultured on M3 (M3M3) medium. ECs exhibited the highest proliferation rate on P3Ar3 medium which was higher than that of M3M3, while the EC proliferation rates of the remaining treatments were lower than that of M3M3. The Ar3Ar3 medium produced the highest numbers of somatic embryos, dicotyledonous embryos, and regenerated plantlets, while the P3P3 medium yielded the fewest somatic embryos and plantlets. Compared with the control, the Put content decreased and the spermine (Spm) content increased in ECs in all other treatments. In addition, the total PA content also increased, except for in the P3Ar3 treatment. The ODC enzyme activities in all treatments were lower than that of the control, while enhanced ADC, DAO, and PAO activities were found in the P3P3, Ar3P3, and Ar3Ar3 treatments, respectively. These results suggest that exogenous Put or D-Arg can stimulate endogenous Put synthesis via regulating enzyme activities and influencing EC proliferation and SE in litchi.

Litchi (Litchi chinensis Sonn.), an important fruit tree in tropical and subtropical regions, possesses substantial economic value. The branch- and leaf-related traits of litchi have a significant impact on litchi yield and quality. However, due to limitations such as the density of the genetic linkage map, there have been few studies on mapping QTLs of branch- and leaf-related traits. In this study, a high-density genetic map was constructed by next-generation sequencing (NGS) using an F1 population of 264 progenies, derived from the cross between the cultivars ‘Sanyuehong’ and ‘Ziniangxi’. A total of 2 574 high-quality BINs (binomial intervals) were obtained, and a genetic linkage map was constructed with a total length of 1 753.3 cM and an average marker distance of 0.68 cM. With the genetic map and the phenotyping of single leaf length (SLL), single leaf width (SLW), leaf shape index (LSI), weight of specific leaf (WSL), petiole length (PL) and compound leaf length (CLL) measured in three seasons, 11, 9, 9, 10, 9 and 12 QTLs were detected for SLL, SLW, WSL, LSI, PL and CLL traits, respectively. Among these QTLs, five QTLs were consistently detected in two seasons and 12 pleiotropic QTLs were identified for at least two traits. These findings will provide new insights for the gene cloning for branch- and leaf-related traits as well as marker-assisted selection (MAS).

Seedless litchi (Litchi chinensis ‘Wu He’) exhibits an excellent parthenocarpic trait, the physiological regulation of which during early fruit development is critical for ultimate yield. Sugar availability serves as the predominant factor governing both fruit abscission and development. However, the molecular mechanisms by which sugar signaling mediates hormonal networks to regulate these processes in seedless litchi remain unclear. This study explored the regulatory patterns in seedless litchi during the massive abscission stage (MA stage) and subsequent developmental stages (SD stages), through integrated physiological and transcriptomic analyses. At the MA stage (15 days after flowering, 15 DAF), the concentrations of glucose and fructose in fruits dropped to their lowest levels. Concurrently, auxin and abscisic acid (ABA) levels increased significantly, while the ethylene (ET) precursor (1-aminocyclopropane-1-carboxylic acid, ACC) content declined. Tissue section revealed that both cell size and cell number were at their lowest, indicating delayed fruit development. Upon entering the SD stages (22 DAF, 29 DAF, 36 DAF), fruit abscission progressively decreased as sugar availability improved. During these stages, cytokinin (iP-type-CK) and gibberellin (GA3) levels increased, accompanied by synchronized increases in cell volume and number, leading to continuous fruit enlargement. Transcriptomic analysis demonstrated that differentially expressed genes were primarily enriched in pathways related to starch and sucrose metabolism, hormone metabolism and signal transduction. During the MA stage, expression of genes such as MAT, ACS, ACO, CTR1, ZEP,NCED, PP2C, and SnRK2 was upregulated, whereas EIN2 was downregulated. In contrast, the SD stages exhibited upregulation of IPT,B-ARR, KAO, GID1, GID2, TFs and DELLA genes, alongside downregulation of CKX, A-ARR, and GA2ox. These gene expression patterns were consistent with fluctuations in hormone levels and collectively regulated fruit abscission and development. RT-qPCR validation confirmed the accuracy of the RNA-Seq results. Deficiencies in fructose and glucose mediate ABA and ET accumulation while disrupting polar auxin transport, thereby exacerbating fruit abscission. Following massive fruit abscission, improved sugar availability enhanced the effects of CK and GA, which promoted cell division and expansion, resulting in gradual fruit enlargement. These findings elucidate the sugar signaling-mediated hormonal regulatory networks during seedless litchi abscission and development, providing a theoretical foundation for achieving high and stable yields in orchards.

Litchi downy blight is a destructive disease that severely reduces yield and causes postharvest losses in litchi fruit. The varieties ‘Heiye’ (HY) and ‘Guiwei’ (GW) were identified as highly resistant and susceptible to Peronophythora litchii infection, respectively. We further investigated the differences between resistant and susceptible varieties through transcriptomic and biochemical analyses to uncover the mechanisms underlying postharvest resistance to litchi downy blight in litchi fruit. RNA-seq analysis of postharvest litchi fruit infected with P. litchii revealed that differentially expressed genes between HY and GW were primarily associated with phenylpropanoid biosynthesis and reactive oxygen species (ROS)-related pathways. The analysis of key metabolites in phenylpropanoid biosynthesis showed a significant difference in lignin content between the two varieties. Fifteen upregulated genes (C4H-1, COMT-1, CAD-6, and POD-7) were identified by RNA-seq and qRT-PCR. These genes correspond to highly active enzymes of C4H, CAD and POD involved in lignin biosynthesis, which were detected in P. litchii-inoculated HY fruit compared to GW. These factors contributed to the higher lignin content in HY fruit. Additionally, the ROS scavenging ability of HY pericarp was superior to that of GW, as evidenced by reduced ROS and malondialdehyde (MDA) levels, increased activities of superoxide dismutase (SOD) and catalase (CAT), and increased expression of two SOD genes and two CAT genes following P. litchii infection. These findings suggest that lignin biosynthesis and ROS metabolism play crucial roles in maintaining litchi fruit resistance to P. litchii, providing valuable insights for breeding and developing resistant varieties.

Pericarp browning of postharvest litchi is a significant obstacle to the industry's high-quality development. Water loss from the pericarp is a key factor triggering browning, but the regulatory mechanism of water metabolism and its relationship with browning remain unclear. In this study, we found that aquaporin activity inhibitors (HgCl2) can delay both water loss and browning in litchi. LcPIP2;4, a PIP family member exhibiting high expression in the litchi pericarp and the greatest water transport activity, is significantly downregulated during water loss and browning. Further analysis revealed that HgCl₂ suppresses both the expression and water transport activity of LcPIP2;4, indicating a close association with the observed browning phenotype. By constructing transient overexpression fruits and transgenic callus tissues of LcPIP2;4 and measuring the water loss rate and browning index, we confirmed that LcPIP2;4 positively regulates water loss and browning in litchi. Through weighted gene co-expression network, LcPIP2;4 promoter sequence and qRT-PCR analysis, we identified 10 potential interacting transcription factors. Yeast one-hybrid, dual-luciferase reporter assay, chromatin immunoprecipitation analysis and electrophoretic mobility shift assay confirmed that LcMYB306 specifically binds to the LcPIP2;4 promoter. In LcMYB306 overexpressing fruits and embryogenic callus, LcPIP2;4 expression was suppressed, resulting in delayed water loss and browning. In contrast, in CRISPR/Cas9-edited LcMYB306 callus, LcPIP2;4 expression was upregulated, and water loss and browning were accelerated, confirming that LcMYB306 negatively regulates this process. This study demonstrates that LcMYB306 delays postharvest water loss and browning in litchi by repressing LcPIP2;4 transcriptionally expression. It provides a theoretical foundation and key target gene for developing litchi varieties resistant to browning.

Litchi, an important tropical fruit, is severely affected by anthracnose disease. However, the mechanism of its disease resistance response remains unknown, and resistant accession genetic resources and resistance-related genes have not yet been identified. In this study, 82 accessions of litchi were evaluated for resistance to Colletotrichum gloeosporioides, and the accessions ‘Haiken 5’ and ‘Nongmei 5 hao’ were identified as resistant and susceptible, respectively. Leaves from these two accessions were inoculated with C. gloeosporioides and collected at 6 and 24 h for use as materials for transcriptome analysis. Analyses of the differentially expressed genes (DEGs) between the accessions and their controls, which were inoculated with potato dextrose agar medium, revealed that the resistant accession presented more DEGs with smaller changes in magnitude, whereas the susceptible accession presented fewer DEGs with greater changes in magnitude. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed, and phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and plant–pathogen interactions were identified as common pathways. Chitinase activity, oxidoreductase activity, aminoglycan and glucosamine-containing compounds, and cell wall metabolic processes also participated in the defence reaction. Salicylic acid signalling in litchi leaves contributed to resistance to C. gloeosporioides. Short Time-series Expression Miner (STEM) and weighted correlation network analysis (WGCNA) were also employed to evaluate the gene expression trends and identify highly correlated genes. Litchi accessions presented different resistance responses to anthracnose disease. Small changes in the expression levels of critical resistance-related genes were sufficient to produce the defence reaction. Calcium ion regulatory mechanisms and transcription factors have been preliminarily identified as contributors to disease resistance. Multiple pathways and molecular processes participate in the defence response. These results identify candidate genes and pathways involved in litchi plant defence against anthracnose.

Genetic breeding and molecular identification in varieties depend on high-performance genotyping tools. The high heterozygosity of the litchi genome contributes to increased resequencing costs and elevated error rates in hybridization-based genotyping methods. In this study, a liquid chip named Litchi40K v1.0 was developed with high-depth resequencing data from 875 litchi samples, and its efficacy was validated across three different populations. In the L. chinensis var. fulvosus population, three subpopulations characterized by spatial distribution, and a total of 1110 genes were identified in the genomic regions with subpopulation differentiation. Additionally, a total of 30 significant signals associated with diverse agronomic traits were identified. The H002 haplotype of LITCHI02696, dominant in the Sub2 subgroup, significantly increased the soluble solid content in the L. chinensis var. fulvosus population. In a hybrid F1 population, a high-density genetic map was constructed and 79 dwarfing-related QTLs were identified with the liquid chip. An NAC transcription factor was identified as a candidate gene with a heterozygous frameshift variant in the male parent. To facilitate the digitization of germplasm resources, 384 SNPs were selected, and the DNA fingerprint map revealed clear genetic relationships and a total of 10 potential synonym groups or instances of bud mutations were identified in 164 main cultivated litchi varieties. This study provides cost-effective, flexible, and versatile liquid chip for genetic analysis and digitalization of germplasm resources in litchi.

Postharvest pericarp browning, caused primarily by the enzymatic oxidation of phenols, reduces the shelf life and market value of litchi fruit and is considered a major limitation for the development of the litchi industry. Previous studies have shown that polyphenol oxidase (PPO) is a key enzyme and that flavonoids are important substrates for enzymatic browning; however, direct evidence is still lacking. This study investigated the differences in the browning process among the wild type (WT) and four PPO gene-edited litchi calli to verify the function of PPO in the browning of litchi tissues. Compared to the WT callus, the proliferation rate, relative expression of litchi PPO gene (LcPPO), PPO activity and color changes significantly decreased or slowed down in all gene-edited calli, indicating that the latter exhibited a slower browning process. Using a liquid chromatography tandem mass spectrometry approach (LC-MS/MS), 83 metabolites of flavonoids were identified, of which 58 were differentially accumulated metabolites (DAMs). Venn analysis revealed 12 common DAMs across different genotypic contrasts that were mostly enriched in the flavonoid biosynthesis pathway. It was presumed that the decrease of LcPPO expression in gene-edited calli led to the reduced PPO activity, then reduced the (-)-epicatechin oxidation. The accumulation of (-)-epicatechin caused the common upregulation of procyanidin B2 and upstream substances such as dihydrokaempferol, taxifolin, naringenin chalcone, 7,4'-dihydroxyflavone, and rutin in their biosynthesis pathways. The results provide novel evidence that (-)-epicatechin acts as the primary direct substrate in the enzymatic browning reaction mediated by PPO.

探索荔枝漆酶(LAC)基因家族的数量、结构和进化关系,可以为解析漆酶基因功能提供理论依据,为探索荔枝果皮褐变机制提供基础.本研究利用生物信息学分析手段,对LAC基因家族成员数量、保守结构域、家族成员的亚细胞定位及染色体位置、系统发育和顺式作用元件,以及基因家族成员在不同组织器官和采后贮藏果皮中的表达情况进行了分析.结果表明,荔枝基因组中共鉴定出61个LAC家族成员,编码的氨基酸残基长度在127～958aa,分子量在14.07～103.47 Da,等电点在4.42～10.34;亚细胞定位分析表明,有19个LAC家族成员定位在叶绿体中,17个LAC家族成员定位在液泡中,其他成员分别定位在内质网、细胞外基质等.61个荔枝LAC家族成员分别定位在10条染色体上,其中第13号和8号染色体上LAC基因数量最多,且呈簇状分布于染色体一端.系统发育树将荔枝LAC家族分为5个亚组,第V亚组最大,有30个家族成员,第Ⅳ亚组最小,仅有8个家族成员.荔枝LAC家族的结构域组织相对保守,具有典型的Cu-oxide漆酶结构域.荔枝LAC基因启动子序列分析发现了大量的顺式作用元件,可能与漆酶家族功能多样性相关.10个荔枝组织器官的转录组测序分析表明,除Lch08g07950-mRNA-1基因外,其余61个基因在荔枝各组织中表达量均较低.实时荧光定量RT-PCR结果与转录组分析结果基本一致,Lch08g07950-mRNA-1在所有组织中都有较高的表达水平,并且其在采后荔枝果皮中的表达水平与褐变发生具有相关性.

Postharvest pericarp browning restricts the shelf life of litchi fruit and was considered as the main bottleneck of litchi industry. Browning is mainly due to the enzymatic catalysis of phenols to form brown substances in the litchi pericarp. (-)-Epicatechin, (-)-gallocatechin, and procyanidin B2 are the main substrates for enzyme catalysis. Previous works have reported that laccase activity was correlated with pericarp browning, and laccase polymerizes substrates in vitro, but the direct evidence was absent. In this research, the sequence of laccase gene LcLac was analyzed, whose expression was correlated with browning index of litchi pericarp at postharvest. Overexpression of LcLac in callus led to higher gene expression and enzymatic activity, and a lower polyphenol content, which contributed to browning of callus. Contrarily, the LcLac gene blocked by CRISPR/Cas9 resulted in an opposite trend of polyphenol contents. LcLac overexpression promoted callus browning, and the gene target block delayed the browning phenomenon. The results clearly indicate that LcLac can be polymerized with polyphenols and is involved in callus browning. LcLac may also participate in polyphenol synthesis and maintain the polyphenol balance in vacuoles.

Litchi (Litchi chinensis Sonn.) is a type of commercially prevalent subtropical and tropical fruit. Since litchi has a highly heterozygous genetic background and a long reproductive cycle, conventional breeding methods (such as hybridization) have limited ability to nurture new litchi cultivars. Here, an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in ‘Feizixiao’ litchi. Transgenic materials were verified using polymerase chain reaction (PCR) analysis, β-glucuronidase (GUS) assay, and green fluorescent protein (GFP) assay. To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology in ‘Feizixiao’ litchi and verify the validity of these transformation systems, the litchi polyphenol oxidase gene (LcPPO, JF926153) was knocked out. Various categories of mutations, covering base insertions, deletions, and substitutions, were found in transgenic materials via sequence analysis. The transformation system achieved high feasibility and efficiency, and the system of CRISPR/Cas9 was successfully employed to edit genes in ‘Feizixiao’ litchi. This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.

Abstract Litchi (Litchi chinensis Sonn.), like other ligneous plants, has long been considered a recalcitrant embryogenic species. Our previous research has shown that adding putrescine (Put) or D-Arginine (D-Arg) to the subculturing medium of embryonic callus (EC) affects EC proliferation and subsequent somatic embryogenesis (SE) in litchi. In this paper, we further confirmed that EC proliferation was significantly increased when either 0.17 mM putrescine (P3) or 2 mM D-Arginine (Ar3) was added to the control medium (M3, MS supplement with 4.52 µM 2,4-D). The subsequent induction of opalescent embryos (OEs) and opalescent dicotyledonous embryos (ODEs) was partially inhibited by Put, and the number of plantlets germinated from the OEs on the P3 medium were lower than those on the M3 medium; however, that was increased by D-Arg. Histomorphological analyses verified various developmental stages of EC proliferation in the M3, P3, and Ar3 media. On the M3 medium, an EC cell was divided into two cells first and then sequentially differentiated through multicell proembryo, globular, heart-shaped, and cotyledonary embryo stages. The EC cells on P3 and Ar3 medium were enlarged more significantly, undergoing repeated cell divisions and then forming a meristematic mass, from which OEs were initiated. The supplementation of Put into the M3 medium promoted the synthesis of endogenous Put and its conversion to Spd and Spm. The PA content in EC on the P3 medium was higher than that on the M3 medium, and the P3 medium enhanced the activity of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and diamine oxidase (DAO); however, it decreased the activity of polyamine oxidase (PAO). The PA content in the Ar3 medium was higher than that in the M3 medium. The supplementation of D-Arg to the M3 medium enhanced ADC and DAO activity but decreased ODC and PAO activity. In the other experiment, EC from the P3 medium was subsequently cultured on M3 (P3M3), P3 (P3P3), and Ar3 (P3Ar3) medium, using EC from M3 medium, and then cultured on M3 (M3M3) medium as a control. The EC proliferation rate of the P3Ar3 treatment was significantly higher than that of the other two treatments. The OEs, ODEs, and plantlets were all most elevated in the P3Ar3 treatment, followed by the P3M3 and P3P3 treatments. EC proliferation and plantlets were significantly higher than in the M3M3 treatment. When ECs were first cultured on Ar3 medium and transferred to M3 (Ar3M3), P3 (Ar3P3), and Ar3 (Ar3Ar3) media for 20 d, the EC proliferation of the Ar3Ar3 treatment was significantly higher than that of the other two treatments. Among the three treatments, EC from the Ar3Ar3 treatment showed the highest OE, ODE, and plantlet induction, followed by the Ar3M3 and P3P3 treatments. The Ar3Ar3 treatment also had the highest induction of OEs, ODEs, and plantlets compared with the other six treatments. EC from the Ar3P3 treatment had the highest Spd, Spm, and PA content, and the M3M3 treatment had the highest Put content. The Spm and PA content of EC from the M3M3 treatment was lower than in the other treatments. Among all treatments, the highest ADC, ODC, DAO, and PAO activity was found in ECs from the M3M3, P3P3, P3P3, and Ar3Ar3 treatments. These results indicate that exogenous Put or D-Arg could stimulate Put synthesis of endogenous Put by regulating the enzyme activities and then affecting the EC proliferation and SE of litchi.

[目的]探究妃子笑荔枝愈伤组织继代及体胚发生过程中结构与多胺变化,为优化荔枝体胚发生技术体系提供理论依据与技术基础.[方法]采用石蜡切片技术、苏木精整体染色法、GC-MS法研究了胚性愈伤组织继代及体胚发生过程中的形态结构及多胺代谢变化.[结果]愈伤组织继代过程中,日增殖量前期增殖缓慢,后期快速增加,达到峰值后下降,最大日增殖量出现在继代后第15天.愈伤组织快速增殖阶段颗粒明显,细胞形态相对均匀,细胞质浓厚,染色深.在体胚发生培养基上约10d时愈伤组织出现明显的厚壁细胞形成生殖隔离,分化原胚,在原胚发生及子叶胚时期有大量淀粉积累.腐胺(Put)和精胺(Spm)是荔枝愈伤组织中多胺(PAs)的主要组分,亚精胺(Spd)含量较低.Put在愈伤组织中维持在较高水平,Put与Spd含量在愈伤组织胚性较强(继代第6天及体胚发生第10天)时都达到最高,体胚发生时愈伤组织中的Spm含量远低于继代时含量.多胺氧化酶(PAO)及二胺氧化酶(DAO)活性与Put及PAs含量呈正相关.[结论]愈伤组织继代约第15天时日增殖量最大,体胚发生过程中约在第10天分化体胚.高的PAO活性与PAs含量有利于荔枝体胚高频发生.

[目的]研究多胺[腐胺(Put)、亚精胺(Spd)、精胺(Spm)]对愈伤组织增殖及体胚诱导的作用,为揭示多胺在荔枝离体再生中的功能提供依据.[方法]采用单因素试验设计,分别在愈伤组织增殖及体胚诱导过程中添加多胺及其合成抑制剂[D-精氨酸(D-Arg)、环己胺(Cha)],比较不同处理下妃子笑荔枝愈伤组织增殖及体胚诱导差异.[结果]低浓度多胺或高浓度多胺合成抑制剂促进愈伤组织增殖,愈伤组织增殖量达显著水平,胚性愈伤组织(EC)与非胚性愈伤组织(NEC)颜色状态界限更加明显.与对照相比,经过多胺处理增殖的愈伤组织乳白胚诱导数及萌发率均降低,而多胺合成抑制剂处理增殖的愈伤组织乳白胚诱导数及萌发率随着多胺合成抑制剂浓度升高而升高,当Cha质量浓度为200 mg·L-1时,乳白胚数量最多,达960个·g-1,萌发率29.41％;当D-Arg质量浓度为700 mg·L-1时,乳白胚萌发率最高,为32.35％,胚数量为450个·g-1.体胚诱导培养基上添加多胺及其合成抑制剂对体胚诱导作用差异较大,Spd促进体胚发生作用较明显,当Spd质量浓度为20mg·L-1时,乳白胚数量为590个·g-1,萌发率为30.30％.体胚发生培养基上添加Cha或高浓度D-Arg抑制体胚发生,而添加低浓度D-Arg则促进体胚发生.[结论]在愈伤组织增殖培养基上添加3种多胺可促进愈伤组织增殖并保持胚性,且抑制原胚发生,而Cha及高浓度D-Arg促进原胚发生.体胚诱导培养基中添加低浓度Spd促进体胚分化.

为开拓荔枝转基因育种新途径,以GUS基因作为报告基因,用花粉管通道法转化授粉后24 h的‘新球蜜荔’荔枝雌花,并统计座果率、成苗率、转化率.结果 显示:共获得303株实生苗,经PCR检测和GUS染色法证实外源基因整合到4株荔枝苗基因组中,转化率为1.32％.此研究结果为荔枝生物技术育种提供了一种简单有效的途径.

为探究荔枝果皮几丁质酶对霜疫霉病抗性中的功能,本研究以抗病品种'黑叶'和感病品种'桂味'为试验材料,分析荔枝果皮中的几丁质酶活性和基因表达情况.结果表明,果皮中几丁质酶活性不同,其与品种的抗病性正相关,霜疫霉菌侵染提高了果皮中酶活性.基于转录组数据,共检测到75条几丁质酶基因,在系统进化树上分为3枝.接种霜疫霉的抗病和感病品种果皮中差异表达基因有58条,表明几丁质酶基因响应霜疫霉入侵表达.不同进化分枝上的3个基因的表达水平因抗病性和接种时间的差异表达模式不同.LcChi3-1基因在抗病品种'黑叶'中的表达水平远远高于感病品种'桂味','黑叶'果皮中LcChi3-1基因的表达水平与酶活性具有显著的相关性,表明其参与了对霜疫霉菌的抗病过程,并发挥主要作用.

采用完全区组设计,研究了不同生长调节剂对'新球蜜荔'体胚发生的影响.结果 表明,在添加TDZ0.5 mg/L、NAA 0.1 mg/L的MS培养基上,'新球蜜荔'体胚高频发生,数量达6 010个/g FW.体胚成熟过程中,PEG-600 25 g/L有一定正向效应,ABA 1 mg/L能改善体胚质量.体胚萌发过程中有少数类似于茎、芽,或弯曲或泡状化的畸形结构,仅有一个体胚抽出茎段,但无真叶展开.石蜡切片观察发现,'新球蜜荔'胚性愈伤组织细胞紧凑,内含物丰富,可见细胞质、细胞核及淀粉粒.不同生长调节剂对体胚发生过程中其愈伤组织形态影响各不相同,ZT、TDZ培养基上体胚发生较KT培养基上的早,ZT培养基上子叶胚大量发生的时间最早.

Copper-containing amine oxidases (CuAOs) have a broad range of functions, including respond to abiotic stresses, formation and differentiation of adipocytes in mammals. To verify the correlation between lipid accumulation and CuAOs in microalgae, the full-length gene of one copper amine oxidase isoform 2 from Chlorella sp. HN11 (ChlCAO2) was cloned, and the gene expression and enzymatic activity were determined. Then the correlation between lipid accumulation and CuAOs activities was further examined in C. vulgaris C3 and Chlamydomonas reinhardtii CC425. The results showed that the cDNA of ChlCAO2 was 4135 bp in length with a 2454 bp open reading frame (ORF). No lipid accumulation and up-regulated ChlCAO2 expression were observed in cells cultured in normal medium (the control) within 96 h. The relative fluorescence intensity of lipids in Chlorella cells under N-starvation was 3.03, 4.48, 4.17, and 4.63 times higher than the control at 24 h, 48 h, 72 h, and 96 h, respectively. While the expression of ChlCAO2 significantly increased by 105, 397, 808, and 333 times higher than the control at 24 h, 48 h, 72 h, and 96 h, respectively, the activity of ChlCAOs of N-starved increased correspondingly. When cells under N-starvation were resupplied with N at 48 h (N-recovery), lipid accumulation, the expression of ChlCAO2 and the activity of ChlCAOs decreased significantly (P < 0.05) at 72 h and 96 h compared with N-starvation. Correlation analysis showed that the relative fluorescence intensity of lipid, the expression of ChlCAO2, and the activity of ChlCAOs in Chlorella were markedly positively correlated with each other. Positive correlation between lipid accumulation and CuAO activities were both observed in C. vulgaris C3 and C.reinhardtii CC425.The results indicated that CuAOs play a key role in the process of lipid accumulation induced by nitrogen starvation in green algae.