The SKP1 gene is an important component of the SCF (SKP1-Cullin1-F-box) complex and serves as a bridge connecting the F-box and Cullin1 genes (F-box-SKP1-Cullin1). The pattern of S-RNase being ubiquitously labelled by the SCF complex and degraded by the 26S protease accounts for the bulk of the available self-incompatibility studies. In this study, 15 ClSKP1s from the ‘Xiangshui’ lemon genome and ubiquitome exist in the same SKP1 conserved domain (CD) as SKP1s in other species. The qPCR results showed that SKP1-6 and SKP1-14 have tissue expression patterns specific for expression in pollen. In addition, SKP1-6 and SKP1-14 in the stigma, style and ovary were significantly upregulated after self-pollination compared to those after cross-pollination. A subcellular location showed that SKP1-6 and SKP1-14 were located in the nucleus. In addition, yeast two-hybrid (Y2H) assays, bimolecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI) assays showed that SKP1-6 interacted with F-box1, F-box33, F-box34, F-box17, F-box19, Cullin1-2 and 26S proteasome subunit 4 homolog A (26S PS4HA). SKP1-14 interacted with F-box17, F-box19, F-box35, Cullin1-2 and 26S PS4HA. The interaction of Cullin1-2 and the F-box with SKP1 as a bridge was verified by a yeast three-hybrid experiment. The ability of S3-RNase to inhibit pollen and pollen tube growth and development was assessed using in vitro pollen co-culture experiments with recombinant S3-RNase proteins. Overall, this study provides important experimental evidence and theoretical basis for understanding the mechanism of self-incompatibility in plants by revealing the key role of the SCF complex in 'Xiangshui' lemon, which is bridged by ClSKP1-6, in self-incompatibility. The results of this study are of great significance for the future in-depth exploration of the molecular mechanism of the SCF complex and its wide application in the self-incompatibility of plants.

SQUAMOSA promoter-binding protein-like (SPL) family genes play an important role in regulating plant flowering and resistance to stress. However, understanding the function of the SPL family in mango is still limited. In a previous study, two MiSPL3 genes, MiSPL3a and MiSPL3b (MiSPL3a/b), were identified in 'SiJiMi' mango and exhibited the highest expression in flowers at the initial flowering stage [24]. Therefore, in this study, we further investigated the expression pattern and gene function of MiSPL3a/b. The results showed that the expression of MiSPL3a was greatest at the end of floral bud differentiation, and MiSPL3b was expressed mainly during the flowering induction and vegetative growth stages. Subcellular localization showed that MiSPL3a/b localized to the nucleus. In addition, ectopic expression of MiSPL3a/b promoted earlier flowering in Arabidopsis thaliana by 3 d-6 d than in wild-type (WT) plants, which increased the expression of SUPPRESSOR OF CONSTANS1 (AtSOC1), FRUITFULL (AtFUL), and APETALA1 (AtAP1). MiSPL3a/b transgenic lines exhibited increased tolerance to drought, GA3, and abscisic acid (ABA) treatments but were sensitive to Pro-Ca treatment. Furthermore, protein interaction analysis revealed that MiSPL3a/b could interact with several stress-related proteins, flowering-related proteins, and the bridge protein 14-3-3. Taken together, MiSPL3a and MiSPL3b acted as positive regulators of flowering time and stress tolerance in transgenic Arabidopsis.

MADS-box genes play a vital role in the vegetative and reproductive growth of plants. In this study, a MiAGL1 gene was cloned and identified from mango (Mangifera indica L.). The DNA sequence of AGAMOUS-LIKE1 (MiAGL1) was 8741 bp in length, including a 723 bp open reading frame and encoding 241 amino acids. MiAGL1 belongs to the MADS-box family. This gene was expressed not only in vegetative tissues but also in floral organs, and the highest expression level was found in flowers. MiAGL1 was expressed in leaves at different floral developmental stages, but the peak appeared at the floral organ differentiation stage. MiAGL1 was present in the cell membrane and nucleus. Ectopic expression of MiAGL1 in Arabidopsis resulted in significant early flowering under long-day conditions. Overexpression of MiAGL1 resulted in abnormal flowering and silique morphology, such as a decrease or absence of petals, smaller petals, and shorter, bent or distorted capsules. The endogenous Arabidopsis thaliana flowering-related genes FT, AP1, and SEP were significantly upregulated, and AtSVP was downregulated in the transgenic lines. Therefore, our data showed that the MiAGL1 gene may play a crucial role in flowering time regulation and floral organ identity.

Flowering promoting factor (FPF) genes play a substantial regulatory role in the process of flowering. In the present study, four MiFPF genes, namely, MiFPF1, MiFPF2, MiFPF3a, and MiFPF3b, were obtained from mango (Mangifera indica L.). Tissue expression analysis showed that MiFPFs were expressed in all mango tissues. Specifically, MiFPF1 and MiFPF2 were highly expressed in leaves, while MiFPF3a and MiFPF3b were highly expressed in flowers and buds. The four MiFPF proteins localize to the nucleus. Overexpression of MiFPFs in transgenic Arabidopsis resulted in early flowering and upregulated the expression of APETAL1 (AP1), FLOWERING LOCUS D (FD) and FLOWERING LOCUS T (FT). MiFPF genes increased the root growth of transgenic Arabidopsis plants under gibberellin treatment. BiFC assays showed that MiFPFs can interact with several DELLA proteins. Taken together, our results demonstrate that the MiFPF gene was involved not only in promoting flowering but also in increasing root growth under gibberellin (GA3) treatment.

14-3-3 genes are universal regulators that play important roles in regulating flower development and stress responses. In this study, two homologous 14-3-3 genes, MiGF6A and MiGF6B, were obtained from the genome of SiJiMi mango. Sequence analysis showed that both MiGF6A and MiGF6B have a fully conserved 14-3-3 super -family domain. MiGF6A and MiGF6B were expressed in tissues at all stages of development, especially in the flowers and buds. Both MiGF6A and MiGF6B were expressed in response to low temperature, NaCl and poly-ethylene glycol (PEG) treatments. Subcellular localization analysis showed that MiGF6A and MiGF6B were located in the nucleus. Overexpression of MiGF6A and MiGF6B in Arabidopsis thaliana resulted in an early -flowering phenotype and significant upregulation of the flowering-related genes FLOWERING LOCUS T (AtFT), AtFD1, and LEAFY (AtLFY). Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses showed that MiGF6A and MiGF6B interact with the MiFD1, MiFD2 and MiSVP3 proteins. These results suggest that MiGF6A and MiGF6B may play positive roles in the mango flowering process.

NAC转录因子是植物中一个大的基因家族,在调控植物生长发育、信号传导、逆境胁迫响应等方面起重要作用.前期研究中,从芒果成花基因酵母文库筛选中分离获得 1 个NAC基因,命名为MiNAC7,本研究对该基因的生物信息学、表达模式和基因功能进行分析.生物信息学分析表明:MiNAC7基因位于 10 号染色体上,有 4 个内含子,5个外显子;MiNAC7基因编码区长度为 1137 bp,编码 379 个氨基酸,理论等电点为 4.88,蛋白质分子量为 93.41 kDa,氨基酸序列中含有 1 个NAM保守结构域.系统进化树分析表明,芒果MiNAC7 与阿月浑子PvNAC26 亲缘关系最接近,同源性最高,氨基酸序列相似性为 69.64%.启动子序列分析显示,MiNAC7基因的启动子区域包含光响应元件、赤霉素响应元件以及生长素响应元件等.表达分析显示,MiNAC7 基因在童期组织的茎与芽中表达水平较高,在叶中表达很低,在成年期组织的茎中表达水平较高,在花和叶中表达很低.同时发现MiNAC7基因在营养生长期的叶片中表达水平较高,在成花转变期和花发育期的叶片中表达水平很低.超量表达的MiNAC7基因诱导转基因拟南芥出现晚花表型,在超量表达MiNAC7基因的拟南芥中显著降低了促花基因AtFT和AtAP1的表达水平,而AtFLC晚花基因的表达水平显著提高.逆境胁迫处理显示,超量表达MiNAC7基因的拟南芥提高了对干旱和盐的抗性,提高了对GA3 的抗性,但对 ABA 更敏感.本研究表明,芒果 MiNAC7 基因不仅影响成花也参与对非生物胁迫的应答,为深入研究 MiNAC7基因参与调控芒果开花和逆境胁迫应答的基因调控网络奠定基础.

Three Di19-4 genes were identified in mango. Overexpression of MiDi19-4B in A. thaliana promoted earlier flowering and enhanced drought, salt, and ABA resistance. Drought-induced protein 19 (Di19) is a drought-induced protein that is mainly involved in multiple stress responses. Here, three Di19-4 genes (MiDi19-4A/B/C) in mango (Mangifera indica L.) were identified, and the coding sequences (CDS) had lengths of 684, 666, and 672 bp and encoded proteins with 228, 222, and 224 amino acids, respectively. The promoters of the MiDi19-4 genes contained phytohormone-, light-, and abiotic stress-responsive elements. The MiDi19-4 genes were expressed in every tissue and highly expressed in leaves. Moreover, MiDi19-4 genes were highly correlated with the vegetative growth period and induced by polyethylene glycol (PEG) or salt stress. MiDi19-4B displayed the highest expression during the vegetative growth period and then showed decreased expression, and MiDi19-4B was highly expressed at both the late stage of the vegetative growth period and the initial stage of the flowering induction period. The 35S::GFP–MiDi19-4B fusion protein was located in the cell nucleus. The transgenic plants ectopically expressing MiDi19-4B exhibited earlier flowering and increased expression patterns of FRUITFULL (AtFUL), APETALA1 (AtAP1), and FLOWERING LOCUS T (AtFT). The drought and salt tolerance of MiDi19-4B transgenic plants was significantly increased, and these plants showed decreased sensitivity to abscisic acid (ABA) and considerably increased expression levels of drought- and salt-related genes and ABA signalling pathway genes. Additionally, bimolecular fluorescence complementation (BiFC) experiments revealed that the MiDi19-4B protein interacted with CAULIFLOWER (MiCAL1), MiCAL2, MiAP1-1, and MiAP1-2. Taken together, these results highlighted the important regulatory roles of MiDi19-4B in tolerance to multiple abiotic stresses and in flowering.

[目的]卵形家族蛋白(ovate family proteins,OFPs)在植物生长发育及逆境响应过程中扮演重要角色.前期通过杧果成花基因酵母文库筛选,获得了一个MiOFP1基因,为明确其功能,对MiOFP1的表达模式和转基因功能开展了研究.[方法]在本研究中分析了杧果MiOFP1的启动子序列;通过实时荧光定量PCR技术分析MiOFP1在杧果不同组织器官和不同生长发育期叶片中的表达模式;转化构建好的超量表达载体并侵染拟南芥研究MiOFP1的功能.[结果]四季蜜杧MiOFP1启动子包含激素响应元件:ABA响应元件、GA响应元件、SA响应元件和乙烯响应元件,逆境响应元件:盐响应元件、脱水响应元件、MYC转录因子和MYB转录因子结合位点.组织特异性表达分析显示,MiOFP1在各组织器官中均有表达,且在童期实生树和成年期嫁接树的茎中表达量最高,在成熟果实中表达量最低;嫁接树不同成花发育时期表达分析结果显示,MiOFP1在营养生长期的叶中表达量最高,在成花诱导期和花发育期表达水平较低.转基因功能研究显示,超量表达MiOFP1的拟南芥出现晚花表型,抽薹期叶片中成花抑制基因FLOWERING LOUS C(FLC)的表达水平显著上调,而成花促进基因FLOWERING LOCUS T(FT)的表达水平显著下调.逆境胁迫处理显示,ABA处理显著抑制拟南芥种子的萌发与根的伸长,但通过转基因显著提高了拟南芥种子的萌发率,降低了拟南芥根长对ABA的敏感性.进一步分析显示,MiOFP1显著提高了拟南芥在ABA处理后的脯氨酸含量和过氧化物酶活性,上调了ABA代谢相关基因的表达水平.[结论]明确了杧果MiOFP1抑制成花,且降低了转基因植株对ABA的敏感性,为进一步探索杧果MiOFP1参与杧果成花和逆境胁迫应答的分子机制奠定基础.

APETALA1 (AP1), CAULIFLOWER (CAL) and FRUITFULL (FUL) were homologous genes with redundant functions in the process of flower transformation and floral development in Arabidopsis. Two CALs genes, MiCAL1 and MiCAL2, were cloned from mango (Mangifera indica L.). Their full-length sequences contained 717 bp and 714 bp, encoding 239 and 238 amino acids, respectively. Both the MiCAL1 and MiCAL2 proteins contained typical MADS-box and K-box domains and therefore belonged to the CAL-like protein family. MiCAL1 and MiCAL2 were expressed in all tissues at the inflorescence elongation stage and flowering stage, with the highest expression in the leaves at the flowering stage. They had similar expression patterns during flower development, with the highest expression levels in leaves during flower differentiation and the lowest expression levels during fruit development. Overexpression of MiCAL1 and MiCAL2 resulted in significantly earlier flowering in Arabidopsis. Overexpression of MiCAL1 resulted in terminal flowers with normal flower organs, while overexpression of MiCAL2 induced partially variation in floral organs but had no effect on inflorescences. Yeast two-hybrid (Y2H) experiments showed that MiCAL1 and MiCAL2 can interact with several flower-related proteins as well as stress response proteins, such as SEP1, SVP1, SVP2, SOC1G and Di19-4. These results suggest that these two MiCALs genes may have an important influence on mango flowering.

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the 'XiangShui' lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in 'XiangShui' lemon are the same as those within other species. Expression pattern analysis revealed that S-3-RNase and S-4-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S-3-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S-1-RNase, S-2-RNase, S-3-RNase and S-4-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S-3-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S-3-RNase interacted with TOPP4. Therefore, S-3-RNase may play an important role in the SI of 'XiangShui' lemon.

Members of the Mi14-3-3 gene family interact with target proteins that are widely involved in plant hormone signal transduction and physiology-related metabolism and play important roles in plant growth, development and stress responses. In this study, 14-3-3s family members are identified by the bioinformatic analysis of the mango (Mangifera indica L.) genome. The gene structures, chromosomal distributions, genetic evolution, and expression patterns of these genes and the physical and chemical properties and conserved motifs of their proteins are analysed systematically. The results identified 16 members of the 14-3-3 genes family in the mango genome. The members were not evenly distributed across the chromosomes, and the gene structure analysis showed that the gene sequence length and intron number varied greatly among the different members. Protein sequence analysis showed that the Mi14-3-3 proteins had similar physical and chemical properties and secondary and tertiary structures, and protein subcellular localization showed that the Mi14-3-3 family proteins were localized to the nucleus. The sequence analysis of the Mi14-3-3s showed that all Mi14-3-3 proteins contain a typical conserved PFAM00244 domain, and promoter sequence analysis showed that the Mi14-3-3 promoters contain multiple hormone-, stress-, and light-responsive cis-regulatory elements. Expression analysis showed that the 14-3-3 genes were expressed in all tissues of mango, but that their expression patterns were different. Drought, salt and low temperature stresses affected the expression levels of 14-3-3 genes, and different 14-3-3 genes had different responses to these stresses. This study provides a reference for further studies on the function and regulation of Mi14-3-3 family members.

【Objective】Xiangshui lemon（Citrus limon（L.） Burm. F.） was used to study the expression of two RHF2A genes, and to screen and verify their interaction proteins by yeast two-hybrid technology and BiFC, so as to lay a foundation for further studying the molecular mechanism of RHF2A in the process of lemon self-incompatibility. 【Method】 Two E3 ubiquitin ligase RHF2A（RING-H2 Zinc Finger2A） genes including RHF2A-1 and RHF2A-2 of Xiangshui lemon were screened from the transcriptome and ubiquitin modification group, and their full-length sequences were cloned. The sequence and protein structure of two RHF2A genes were analyzed by bioinformatics to predict the cis acting elements of their promoters. 35S-RHF2A-GFP fusion protein expression vector was constructed for subcellular localization analysis. The temporal and spatial expression patterns of two RHF2A were analyzed by real-time fluorescence quantitative PCR. The yeast two-hybrid bait vector was constructed to screen the interaction proteins from the lemon yeast library. The BiFC vector was constructed to verify the interaction of the target protein in onion living cells.【Result】The RHF2A-1 and RHF2A-2 genes were obtained from ‘Xiangshui’ lemon, and the total length of ORF was 1 161 and 1 134 bp, respectively. NCBI domain prediction found that it had a Ring/U-box domain. Promoter analysis showed that there were POLLEN1LELAT52 and GTGANTG10 related to pollen specific expression elements. Tissue expression analysis showed that RHF2A-1 gene was specifically expressed in pollen and RHF2A-2 was specifically expressed in leaves; the results of temporal and spatial expression analysis showed that the expressed of RHF2A-1 in self-stigma tended to increase from the first day and reached the peak on the third day, which was more than 5 times that of hybrid stigma. Subcellular localization showed that RHF2A-1and RHF2A-2 were localized in the nucleus. The interaction protein predicted by Uniprot website showed that RHF2A could interact with KRP6, AT3G57370, UBA1, FBL17 and SK11proteins, and RHF2A gene was involved in biological processes such as self-incompatibility ubiquitination reaction pathway, gametophyte development regulation and pollen growth and development. 72clones were screened by yeast two-hybrid technology. After sequencing and blast comparison, the repetitive clones were excluded.Finally, 20 candidate interaction proteins such as ABCF3 were obtained. Through one-to-one interaction verification and BiFC, it was determined that there was an interaction relationship between RHF2A-1 and ABCF3-2. 【Conclusion】 The temporal and spatial expression of RHF2A-1 gene was consistent with the germination of pollen on pistil in the process of self-incompatibility; the interaction candidate proteins directly affecting pollen growth and development during the pollination were screened, which preliminarily proved that RHF2A-1 gene played an important role in the process of self-incompatibility.

SQUAMOSA promoter binding protein-like (SPL) genes played important roles during plant growth and development. However, there have been no studies on SPL genes in mango (Mangifera indica L.). In the present study, a total of 26 SPL family members were first identified and analysed in the ‘SiJiMi’ mango genome, and these genes were classified into nine subfamilies (G1 - G9) according to conserved gene structures and protein motifs. Expression analysis revealed that the MiSPL genes were expressed in all the tested organs, albeit with expression level differences. Fifteen of them exhibited the highest expression in flowers at the initial flowering stage. Furthermore, most of the MiSPLs were expressed in response to exogenous gibberellin (GA3) and prohexadione-calcium (Pro-Ca) treatments. MiSPL13 was primarily expressed in flowers and significantly upregulated in buds under GA3 and Pro-Ca treatments on the 2nd and 6th days, respectively. The full length cDNA sequence of MiSPL13 was 1116 bp, encoding 372 amino acids. MiSPL13 was located in the nucleus and had transcriptional activation activity in yeast. Overexpression of MiSPL13 not only promoted early flowering in transgenic Arabidopsis, which increased the expression levels of AtAP1, AtSOC1, and AtFUL, but also significantly increased tolerance to drought, abscisic acid (ABA) and GA3 while showing sensitivity to Pro-Ca treatment.

[目的]了解沃柑叶片响应柑橘溃疡病侵染的分子机制及易感品种与柑橘溃疡病菌的互作应答机制,筛选出柑橘溃疡病菌危害沃柑叶片时的相关应答基因,为抗性育种提供基因基础.[方法]以接种溃疡病菌后0、2、4、6和8 d的沃柑叶片为试材,利用Novaseq 6000平台进行转录组双向测序,并对数据进行生物信息学相关性分析.[结果]接种无菌水(CK)后0、2、4、6和8d获得的Cleanreads分别是48 482 127、47 270 288、50 998 549、53 201 972和47 924 731 条,接种柑橘溃疡病菌(JZY)后 0、2、4、6 和 8 d获得的 Clean reads 分别是 51 042 967、49 552 248、46 734 029、47 940 345 和45 371 891条.接种后0、2、4、6和8 d的上调差异表达基因数量分别为1、947、1081、656和2108个,下调差异表达基因数量分别为1、343、753、303和1908个.在接种后2、4、6和8 d有374个基因均表达差异,其中上调基因为61个,下调基因为313个.GO功能富集分析结果显示,沃柑叶片响应溃疡病不同时期的差异表达基因,主要集中在生物进程、分子功能和细胞组分中.KEGG注释分析结果显示,差异表达基因主要参与次生代谢物的生物合成、植物与病原体相互作用、植物激素信号转导、过氧化物酶体和蛋白质内质网合成等途径.[结论]植物与病原体相互作用、植物激素信号转导、过氧化物酶体和蛋白质内质网合成4条通路为柑橘感抗病相关的重要代谢通路.研究结果可作为深入研究柑橘种质资源感抗溃疡病基因和探究柑橘与病原菌互作分子机制的理论提供参考依据.