摘要：【目的】深入分析蘋果sMdCAX11（去掉NRR區(qū)域的MdCAX11）基因功能?！痉椒ā糠謩e利用蜜脆蘋果果實(shí)和擬南芥材料，采用過表達(dá)sMdCAX11的試驗(yàn)方法，觀察果實(shí)、葉片等各組織的表型，并測(cè)定不同組織的礦質(zhì)元素含量，同時(shí)對(duì)蘋果MdCAX11基因啟動(dòng)子區(qū)域進(jìn)行預(yù)測(cè)分析以及啟動(dòng)子轉(zhuǎn)錄活性分析?！窘Y(jié)果】瞬時(shí)過表達(dá)sMdCAX11的蘋果果肉顏色變褐，并出現(xiàn)皺縮；同時(shí)過表達(dá)sMdCAX11的蘋果果肉和擬南芥葉片的總Ca含量明顯下降，且元素比值（K+Mg）/Ca明顯升高。【結(jié)論】sMdCAX11基因過表達(dá)可導(dǎo)致植株組織的元素分配不均，不同元素間的比例失衡。

關(guān)鍵詞：蘋果；Ca2+/H+反向轉(zhuǎn)運(yùn)體（CAX）；基因功能；礦質(zhì)元素；鈣含量

中圖分類號(hào)：S661.1文獻(xiàn)標(biāo)志碼：A文章編號(hào)：1009-9980（2024）07-1275-10

Functional analysis and characterization of the sMdCAX11 gene in apple

LIU Jia1，2，YIN Weijie2，LI Yukun3，WANG Caixia1*，REN Xiaolin2*

（1Institute of Chinese Materia Medica，China Academy of Chinese Medical Sciences，Beijing 100700，China;2College of Horticulture，Northwest Agriculture and Forestry University，Yangling 712100，Shaanxi，China;3Mengniu Hi-tech Dairy Product Beijing Co.，Ltd.，Bei-jing 101100，China）

Abstract：【Objective】Ca2+/H+antiporter（CAX）is a type of low-affinity and high-capacity transporter that mainly relies on the transmembrane proton gradient to complete the transport of Ca2+.This protein may be related to the occurrence of plant calcium deficiency.It is known that Ca2+/H+reverse transport-er proteins（CAXs）in model plants like Arabidopsis thaliana and tomato play important roles in regulat-ing intracellular Ca2+distribution and allocation，and maintaining intracellular calcium homeostasis，and overexpression of the CAXs genes in different plants could cause calcium deficiency symptoms in the plants.In the preliminary stage of this study，Honeycrisp apple bitter pit disorder fruits with different de-grees of incidence were used as test materials，and the differences in mineral element contents and ex-pression patterns of calcium transport-related genes in disordered fruits were analyzed.The key regula-tory genes MdCAX5 and sMdCAX11（MdCAX11 with the N-terminal autoinhibitory region removed）in-volved in the development of bitter pit disorder were then identified.However，the function of Md-CAX11 protein in apple was still unclear.Meanwhile，it was still unclear whether Ca2+/H+reverse trans-porter proteins were involved in the development of bitter pit disorder in fruit.【Methods】The gene functions of sMdCAX11 were analyzed using experimental methods like genetic transformation.In this study，we first utilized the transient transformation of Honeycrisp apple fruits to verify the calcium trans-port function of the sMdCAX11 protein.Fruits transiently transformed with the sMdCAX11 gene were sectioned to observe the changes in the flesh tissue near the injection hole，and the mineral element con-tent of the flesh tissue was also determined.Next，we stably transformed sMdCAX11 into the Arabidop-sis Col-0 and successfully obtained positive T4 generation transgenic plants.The PCR tests at DNA lev-el and RNA level verified that all obtained were positive plants，and the leaves were analyzed for miner-al element detection.In this study，various types of elements contained in the 1500 bp promoter region upstream of the start codon of the apple MdCAX11 gene were also predicted and analyzed.Meanwhile，in order to investigate the effect of the ProCAX11 promoter on the response to Ca2+，tobacco leaves sprayed with different concentrations（0，10，20 and 40 mmol·L-1）of CaCl2 were infiltrated by using Agrobacterium proCAX11：：GUS.The effect of calcium ion on the transcriptional activity of MdCAX11 gene promoter was verified by GUS staining and GUS protein activity analysis.【Results】The total cal-cium content in apple flesh tissues overexpressing the sMdCAX11 gene significantly decreased and con-tinued to decrease with the extension of storage time.By analyzing the total Mg and K contents in the flesh tissues，these two elements showed a rapid increase after a transient decrease at the 3rd day after infestation，reaching the highest value at the 5th day.Further analysis of the elemental ratios of the flesh tissues revealed that the total mineral elements（K+Mg）/Ca significantly increased in the flesh of transiently transformed sMdCAX11 and continued to rise with the extension of storage time.At the 5th day of infestation，（K+Mg）/Ca ratio of water-soluble mineral elements was significantly higher in the flesh tissues of transiently transformed sMdCAX11 genes，while there was no significant change in the elemental ratios of the control.By analyzing the leaf mineral element contents of the four sMdCAX11 transgenic Arabidopsis lines，consistent with the apple flesh material transiently transformed with sMd-CAX11，the（K+Mg）/Ca ratios of the total elements，and the ratios of the water-soluble elements ap-peared to be marked increase and significantly different in the positive plants.A large number of cis-acting elements responsive to external environmental conditions were present in the promoter region of the MdCAX11，such as ARE，an element involved in anaerobic induction，as well as the light-respon-sive elements ATCT-motif，Box 4，G-box，GT1-motif，TCCC-motif and chs-CMA2a.In addition，the MdCAX11 promoter region was characterized by the presence of several transcription factor binding sites，such as the WRKY transcription factor binding sites WBOXNTERF3，WBOXATNPR1 and WRKY710S，as well as the binding sites of transcription factors like MYB.By analyzing the GUS pro-tein activity of tobacco leaves，it was found that ProCAX11 initiation significantly increased in a high calcium environment.The GUS protein activity significantly increased when they were sprayed with different concentrations of CaCl2，and the difference was significant compared with the control.Simul-taniously，the GUS protein activity reached the highest value when they were sprayed with 20 mmol·L-1 CaCl2.The transcriptional activity of MdCAX11 promoter was significantly affected by Ca2+.【Conclu-sion】The calcium transport capacity of the MdCAX11 gene was influenced by the N-terminal autoin-hibitory region，and the sMdCAX11 gene was equipped to transport calcium ions.Overexpression of the sMdCAX11 gene significantly reduced the calcium content in plants and disrupted the balance of mineral element ratios.sMdCAX11 transgenic Arabidopsis thaliana leaves contained significantly lower total Ca content as well as water-soluble Ca content compared with the wild type，and the（K+Mg）/Ca ratio of the total and water-soluble mineral elements was significantly higher than that of the control.In conclusion，these findings provided further evidence that overexpression of the sMdCAX11 gene can cause uneven distribution of elements in plant tissues and imbalance in element proportions.

Keywords：Apple;Ca2+/H+antiporter（CAX）;Gene function;Mineral elements;Calcium content

Ca2+/H+反向轉(zhuǎn)運(yùn)體（CAXs）是一類低親合、高容量的轉(zhuǎn)運(yùn)蛋白，即使胞質(zhì)中Ca2+水平較低時(shí)也可以發(fā)揮其生理功能。近年來，在擬南芥、水稻、葡萄等植物中已克隆出多個(gè)CAX基因，這些基因編碼的蛋白不僅可以運(yùn)輸Ca2+，還能運(yùn)輸Mn2+[1-2]。將擬南芥的AtCAX4基因超表達(dá)載體轉(zhuǎn)入到番茄中，獲得轉(zhuǎn)基因番茄苗，其中轉(zhuǎn)基因植株對(duì)鈣離子的吸收增強(qiáng)，植株體內(nèi)的鈣元素含量增加，甚至延長(zhǎng)果實(shí)的貨架期[3]。

研究CAXs的蛋白結(jié)構(gòu)發(fā)現(xiàn)，幾乎所有的Ca2+/H+反向轉(zhuǎn)運(yùn)蛋白都有著相似的結(jié)構(gòu)（圖1），400 aa左右的氨基酸，均含有11個(gè)跨膜區(qū)域（TMD），都存在著一個(gè)Ca2+的結(jié)合區(qū)（CaD），以此調(diào)節(jié)CAXs蛋白轉(zhuǎn)運(yùn)鈣離子的能力[4]。Ca2+/H+反向轉(zhuǎn)運(yùn)蛋白的N端有親水性自抑制區(qū)（NRR），N末端自抑制區(qū)存在于細(xì)胞質(zhì)中，該段序列可直接影響CAX蛋白的活性[5]。利用酵母突變體K667菌株進(jìn)行功能互補(bǔ)實(shí)驗(yàn)時(shí)發(fā)現(xiàn)，Ca2+的轉(zhuǎn)運(yùn)受CAXs蛋白N-末端自抑制區(qū)的調(diào)控，全長(zhǎng)的CAXs基因編碼的蛋白無法有效地轉(zhuǎn)運(yùn)Ca2+[6-7]。CAXs的這種轉(zhuǎn)運(yùn)特性存在于大多數(shù)植物的Ca2+/H+反向轉(zhuǎn)運(yùn)蛋白中，例如擬南芥AtCAX1、AtCAX2、AtCAX3、AtCAX4[8]，棉花GhCAX1、GhCAX3[9]，水稻OsCAX1、OsCAX3和OsCAX4[10]。

蛋白的調(diào)控機(jī)制在不同植物品種及不同的成員之間不盡相同。例如，綠豆中去NRR的sVCAX1可以彌補(bǔ)K667酵母菌株轉(zhuǎn)運(yùn)Ca2+的缺陷，同時(shí)轉(zhuǎn)化全長(zhǎng)MdCAX2L-2的K667菌株也可以正常轉(zhuǎn)運(yùn)Ca2+[11]。相似的情況也多有報(bào)道，白菜全長(zhǎng)BrCAX蛋白和蘋果全長(zhǎng)MdCAX5蛋白在酵母菌株K667中均可彌補(bǔ)突變株的鈣轉(zhuǎn)運(yùn)缺陷[12-13]。研究去除N-末端自抑制區(qū)sPutCAX1的基因功能時(shí)發(fā)現(xiàn)，與星星草的全長(zhǎng)PutCAX1基因相比，sPutCAX1的鈣轉(zhuǎn)運(yùn)能力明顯下降[14]。同時(shí)也有文獻(xiàn)表明，CAXs的N-末端自抑制區(qū)在不同的植物體細(xì)胞中有各自不同的調(diào)節(jié)功能，但目前對(duì)這些相關(guān)調(diào)節(jié)機(jī)制尚不明了。通過對(duì)CAXs蛋白多樣性的研究可以發(fā)現(xiàn)，不同物種之間的CAXs蛋白存在著較大的差別。

在前期研究中發(fā)現(xiàn)sMdCAX11（去NRR的Md-CAX11）也表現(xiàn)出較強(qiáng)的鈣轉(zhuǎn)運(yùn)能力，與MdCAX5的基因功能相似[12]。同時(shí)sMdCAX11蛋白作為一價(jià)陽離子和二價(jià)陽離子的轉(zhuǎn)運(yùn)體，既可以轉(zhuǎn)運(yùn)Ca2+，也可以轉(zhuǎn)運(yùn)Na+。因此在本研究中將重點(diǎn)研究sMdCAX11的基因功能，通過觀察過表達(dá)sMdCAX11試驗(yàn)材料的表型及分析不同組織的礦質(zhì)元素含量，研究sMd-CAX11蛋白在植物體內(nèi)所起到的關(guān)鍵作用。

1材料和方法

1.1試驗(yàn)材料

用于瞬時(shí)轉(zhuǎn)化的蜜脆蘋果果實(shí)采收自陜西省寶雞市西北農(nóng)林科技大學(xué)千陽試驗(yàn)站，選取無病害、機(jī)械損傷的蘋果，樣品采集后迅速帶回實(shí)驗(yàn)室，1℃貯藏冷庫(kù)存放。用于瞬時(shí)轉(zhuǎn)化的本氏煙草放置于光照培養(yǎng)箱進(jìn)行培養(yǎng)（培養(yǎng)條件：22℃/20℃，16 h光照/8 h黑暗），培養(yǎng)至6～8枚葉時(shí)用于試驗(yàn)。用于穩(wěn)定轉(zhuǎn)化的擬南芥為Col-0生態(tài)型，光照培養(yǎng)條件為16h光照（22℃）和8 h黑暗（20℃）。

克隆載體pMD19-T Simple vector購(gòu)自TaKaRa公司，植物表達(dá)載體pVBG2307、pC0390GUS等均由實(shí)驗(yàn)室保存。大腸桿菌E.coli DH5“購(gòu)自天根公司，農(nóng)桿菌菌株GV3101感受態(tài)購(gòu)自上海唯地生物有限公司。

1.2蘋果的瞬時(shí)轉(zhuǎn)化

克隆sMdCAX11基因CDS序列（去掉終止密碼子），將得到的片段插入到融合載體GFP蛋白的N端，得到新的融合載體35S：：sMdCAX11-GFP。將獲得的融合載體通過凍融法轉(zhuǎn)入農(nóng)桿菌GV3101感受態(tài)細(xì)胞，獲得陽性農(nóng)桿菌。蘋果瞬時(shí)轉(zhuǎn)化的方法參考Jiang等[15]方法進(jìn)行，在果實(shí)瞬時(shí)轉(zhuǎn)化的第3天、第5天、第9天采樣并液氮速凍后保存于-80℃冰箱。

1.3擬南芥的穩(wěn)定轉(zhuǎn)化方法

利用方法1.2中獲得的含有35S：：sMdCAX11-GFP的農(nóng)桿菌用于擬南芥的穩(wěn)定轉(zhuǎn)化，采用浸花序法獲得陽性擬南芥植株。

1.4總Ca、Mg、K、N和P含量的測(cè)定

1.4.1總Ca、Mg和K含量的測(cè)定稱取3.00 g果肉凍樣置于70℃烘箱中烘至恒質(zhì)量，稱取1.00 g烘干樣品并放置于100mL消解管中，同時(shí)加入3mL高氯酸和12mL硝酸，浸泡過夜后進(jìn)行高溫消解，對(duì)消解樣品趕酸、定容后稀釋一定倍數(shù)，利用原子吸收光譜儀（ZA3000）測(cè)定樣品的總Ca、K和Mg含量。

1.4.2總N和P含量的測(cè)定稱取0.20 g烘干樣品與8 mL硫酸混合后放入100 mL消解管中浸泡過夜，經(jīng)高溫消解、趕酸、定容、稀釋后利用連續(xù)流動(dòng)化學(xué)分析儀測(cè)定總N、P含量。

總礦質(zhì)元素含量以干質(zhì)量表示，每項(xiàng)測(cè)定均包括3次生物學(xué)重復(fù)。

1.5水溶性Ca、Mg、K、N和P含量的測(cè)定

水溶性礦質(zhì)元素的測(cè)定方法參照Pavicic等[16]的報(bào)道并有所改動(dòng)。稱取6 g凍樣置于研缽中，加20 mL去離子水充分研磨，將研磨后的勻漿10 000r·min-1離心30min。收集上清液，將離心管的沉淀用20 mL去離子水重懸后，如上所述再次離心。收集兩次離心后的上清液經(jīng)多次濾紙過濾后定容到50 mL，稀釋至一定倍數(shù)后利用原子吸收光譜儀測(cè)定水溶性Ca、Mg和K含量，利用連續(xù)流動(dòng)化學(xué)分析儀測(cè)定水溶性N和P含量。水溶性礦質(zhì)養(yǎng)分含量以鮮質(zhì)量表示。每項(xiàng)測(cè)定均包括3次生物學(xué)重復(fù)。

1.6 ProCAX11啟動(dòng)子的克隆及順式作用元件分析

采用植物基因組DNA提取試劑盒（AG21011）提取植物總DNA。以MdCAX11全長(zhǎng)在蘋果基因組數(shù)據(jù)庫(kù)中比對(duì)，獲得起始密碼子上游1500bp左右的核苷酸序列。隨后設(shè)計(jì)引物，以蜜脆葉片DNA為模板，克隆MdCAX11基因啟動(dòng)子序列。利用Plant CARE和PLACE在線網(wǎng)站預(yù)測(cè)ProCAX11啟動(dòng)子存在的轉(zhuǎn)錄因子結(jié)合位點(diǎn)及順式作用元件。

1.7 GUS染色方法及蛋白活性分析

克隆MdCAX11基因啟動(dòng)子ProCAX11序列，將得到的片段插入到載體pC0390GUS，得到新的融合載體ProCAX11：：GUS，對(duì)照為pC0390GUS空載。將獲得的融合載體采用凍融法轉(zhuǎn)入農(nóng)桿菌GV3101感受態(tài)細(xì)胞。PCR鑒定陽性的菌液瞬時(shí)侵染本氏煙草。GUS染色、GUS粗蛋白提取及濃度測(cè)定、GUS蛋白熒光值測(cè)定等方法參照Chen等[17]的報(bào)道。

1.8 ProCAX11啟動(dòng)子對(duì)不同濃度CaCl2的響應(yīng)分析

為了探究ProCAX11啟動(dòng)子對(duì)CaCl2的響應(yīng)效果，配制濃度為0、10、20和40 mmol·L-1的CaCl2溶液。在侵染前24h時(shí)，對(duì)本氏煙草植株噴灑不同濃度的CaCl2溶液，葉片的正反面均勻噴灑，直至葉片兩面均被打濕且不斷滴水為止，然后放回原來的培養(yǎng)條件下繼續(xù)培養(yǎng)。利用轉(zhuǎn)化有融合載體Pro-CAX5：：GUS的農(nóng)桿菌侵染煙草葉片。侵染48 h后對(duì)侵染的煙草葉片進(jìn)行GUS染色及GUS蛋白活性分析。

2結(jié)果與分析

2.1 sMdCAX11瞬時(shí)過表達(dá)在蘋果果實(shí)的表型鑒定及元素分析

利用瞬時(shí)轉(zhuǎn)化蜜脆蘋果果實(shí)的方法，來驗(yàn)證sMdCAX11蛋白的鈣轉(zhuǎn)運(yùn)功能。對(duì)瞬時(shí)轉(zhuǎn)化sMd-CAX11基因的果實(shí)進(jìn)行切片，觀察注射孔附近的果肉組織，發(fā)現(xiàn)在侵染第9天時(shí)果肉組織明顯變褐，果肉組織皺縮，與對(duì)照組相比差異顯著（圖2-A）。基因相對(duì)表達(dá)量分析檢測(cè)侵染第9天時(shí)果肉的sMd-CAX11基因表達(dá)量顯著上調(diào)，這也直接說明了瞬時(shí)轉(zhuǎn)化試驗(yàn)效果良好，可以用于進(jìn)一步的分析檢測(cè)（圖2-B）。

2.2 sMdCAX11瞬時(shí)過表達(dá)在蘋果果實(shí)的元素分析

分析瞬時(shí)轉(zhuǎn)化sMdCAX11基因的果肉組織總礦質(zhì)元素及水溶性礦質(zhì)元素的含量，對(duì)照組為瞬時(shí)轉(zhuǎn)化空載的果肉組織，結(jié)果發(fā)現(xiàn)過表達(dá)sMdCAX11基因的果肉組織總鈣含量顯著下降，且隨著貯藏時(shí)間的延長(zhǎng)而不斷降低（圖3-A）。在侵染后第3天時(shí)，水溶性Ca含量與對(duì)照組相比出現(xiàn)了短暫的上升，但隨著貯藏時(shí)間的延長(zhǎng)而顯著下降（圖3-B）。分析果肉組織的總Mg、K含量，這兩種元素在侵染后第3天時(shí)出現(xiàn)短暫降低之后迅速升高，在第5天時(shí)達(dá)到最高值。而水溶性Mg、K含量則與總Mg、K含量的變化趨勢(shì)相反（圖3）。進(jìn)一步分析果肉組織的元素比值發(fā)現(xiàn)，總礦質(zhì)元素（K+Mg）/Ca比值在瞬時(shí)轉(zhuǎn)化sMdCAX11的果肉中顯著增加，且隨著貯藏時(shí)間的延長(zhǎng)而不斷升高。在侵染第5天時(shí)，水溶性礦質(zhì)元素的（K+Mg）/Ca比值在瞬時(shí)轉(zhuǎn)化sMdCAX11基因的果肉組織中明顯升高，同時(shí)對(duì)照組的元素比值無顯著變化（圖3）。

2.3過表達(dá)sMdCAX11擬南芥的元素分析

為進(jìn)一步驗(yàn)證sMdCAX11參與鈣轉(zhuǎn)運(yùn)的功能，將sMdCAX11穩(wěn)定轉(zhuǎn)化擬南芥Col-0生態(tài)型，并成功獲得陽性T4代轉(zhuǎn)基因植株。通過DNA水平及RNA水平的PCR檢驗(yàn)，驗(yàn)證所獲得的均為陽性植株，并開展后續(xù)試驗(yàn)。對(duì)擬南芥葉片進(jìn)行礦質(zhì)元素檢測(cè)分析，結(jié)果發(fā)現(xiàn)4個(gè)過表達(dá)sMdCAX11擬南芥株系的葉片總Ca含量與水溶性Ca含量均出現(xiàn)顯著下降，差異極顯著（圖4）。同時(shí)4個(gè)株系的陽性樣本間差異不顯著，說明轉(zhuǎn)基因植株間的表型穩(wěn)定，不存在特異性。分析4個(gè)轉(zhuǎn)基因擬南芥株系葉片的總Mg與水溶性Mg含量，發(fā)現(xiàn)與野生型對(duì)照組相比差異不顯著（圖4）。然而4個(gè)轉(zhuǎn)基因擬南芥株系的總K含量與水溶性K含量均高于野生型且差異顯著，但在4個(gè)株系間差異不顯著（圖4）。與瞬時(shí)轉(zhuǎn)化sMd-CAX11的蘋果果肉材料相一致的是元素的（K+Mg）/Ca比值，總元素的比值與水溶性元素的比值在陽性植株中均出現(xiàn)明顯增大，且差異顯著（圖4）。

2.4 ProCAX11啟動(dòng)子順式作用元件分析

利用Plant CARE等在線網(wǎng)站，對(duì)蘋果Md-CAX11基因起始密碼子上游1500bp啟動(dòng)子區(qū)域所包含的各類元件進(jìn)行預(yù)測(cè)分析（表1）。結(jié)果表明，該基因啟動(dòng)子區(qū)存在大量響應(yīng)外界環(huán)境條件的順式作用元件，如參與厭氧誘導(dǎo)的元件ARE，以及參與光響應(yīng)的元件ATCT-motif、Box 4、G-box、GT1-motif、TCCC-motif和chs-CMA2a（表1）。該基因的啟動(dòng)子也存在參與激素應(yīng)答的調(diào)控元件，如參與脫落酸的ABRE（表1）。另外，ProCAX11啟動(dòng)子區(qū)域還存在著多個(gè)轉(zhuǎn)錄因子結(jié)合位點(diǎn)，如WRKY轉(zhuǎn)錄因子結(jié)合位點(diǎn)WBOXNTERF3、WBOXATNPR1和WRKY710S，以及MYB等轉(zhuǎn)錄因子的結(jié)合位點(diǎn)（表1）。

2.5 ProCAX11啟動(dòng)子轉(zhuǎn)錄活性及鈣元素響應(yīng)分析

為探究ProCAX11啟動(dòng)子對(duì)CaCl2的響應(yīng)效果，利用轉(zhuǎn)化融合載體ProCAX11：：GUS的農(nóng)桿菌侵染噴灑過不同濃度（0、10、20、40 mmol·L-1）CaCl2的煙草葉片，并采用GUS染色及GUS蛋白活性分析的方法來驗(yàn)證鈣離子對(duì)MdCAX11基因啟動(dòng)子轉(zhuǎn)錄活性的影響（圖5）。GUS染色發(fā)現(xiàn)噴灑10 mmol·L-1與20 mmol·L-1 CaCl2的煙草葉片顏色相比于其他組明顯更深，CK為注射空載農(nóng)桿菌煙草葉片（圖5-A）。通過分析煙草葉片的GUS蛋白活性，發(fā)現(xiàn)在高鈣的環(huán)境下MdCAX11啟動(dòng)效果明顯增強(qiáng)。在噴灑不同濃度的CaCl2時(shí)，GUS蛋白活性顯著提高，與對(duì)照組相比差異顯著。同時(shí)在噴灑20 mmol·L-1 CaCl2時(shí)，GUS蛋白活性達(dá)到最高值（圖5-B）。ProCAX11啟動(dòng)子轉(zhuǎn)錄活性受Ca2+的顯著影響。然而，隨著鈣離子濃度的增加，GUS蛋白的活性并沒有隨之增高（圖5）。

3討論

在前期研究中已經(jīng)證實(shí)sMdCAX11（去NRR的MdCAX11）可以表現(xiàn)出較強(qiáng)的鈣轉(zhuǎn)運(yùn)能力[12]。同時(shí)sMdCAX11蛋白作為一價(jià)陽離子和二價(jià)陽離子的轉(zhuǎn)運(yùn)體，既可以轉(zhuǎn)運(yùn)Ca2+，也可以轉(zhuǎn)運(yùn)Na+。為驗(yàn)證sMdCAX11蛋白的功能，筆者在本研究中分別利用了蘋果果實(shí)和擬南芥材料，采用瞬時(shí)過表達(dá)和穩(wěn)定過表達(dá)sMdCAX11的試驗(yàn)手段，觀察果肉組織的表型，并測(cè)定不同組織的礦質(zhì)元素含量。

在白菜[13]、番茄[5]和土豆[18]中過表達(dá)液泡CAX轉(zhuǎn)運(yùn)蛋白，植株出現(xiàn)了類似缺鈣的癥狀。瞬時(shí)過表達(dá)sMdCAX11的蘋果在侵染第9天時(shí)注射孔附近的果肉顏色變褐，并出現(xiàn)皺縮，有明顯的死細(xì)胞，而這一癥狀與苦痘病發(fā)病部位的果肉表型十分相像。分析瞬時(shí)轉(zhuǎn)化sMdCAX11基因的果肉組織總礦質(zhì)元素及水溶性礦質(zhì)元素的含量。過表達(dá)sMdCAX11基因的果肉組織相比于對(duì)照組總鈣含量顯著下降。分析果肉組織的元素比值發(fā)現(xiàn)，總礦質(zhì)元素及水溶性礦質(zhì)元素（K+Mg）/Ca比值在瞬時(shí)轉(zhuǎn)化sMdCAX11的果肉中顯著增大，且隨著貯藏時(shí)間的延長(zhǎng)而不斷升高。這一結(jié)果與苦痘病果實(shí)中不同礦質(zhì)元素的分布及比例極其相似?？喽徊」麑?shí)中的水溶性Ca含量顯著低于健康果實(shí)，且（K+Mg）/Ca比值顯著高于對(duì)照果實(shí)[19-20]。

穩(wěn)定過表達(dá)sMdCAX11的擬南芥葉片在礦質(zhì)元素含量及比值的檢測(cè)結(jié)果上與蘋果果實(shí)相一致，總Ca含量與水溶性Ca含量在陽性植株的葉片中明顯下降，且（K+Mg）/Ca比值在陽性植株中顯著升高。這一結(jié)果也證實(shí)了sMdCAX11基因過表達(dá)可導(dǎo)致植株組織的元素分配不均，不同元素間的比例失衡的結(jié)論。但這一結(jié)果與先前研究報(bào)道并不完全相符[21]。這可能是因?yàn)楣P者在本研究中對(duì)果實(shí)的Ca含量檢測(cè)時(shí)并未對(duì)細(xì)胞膜、細(xì)胞質(zhì)等分別進(jìn)行檢測(cè)，所以Ca含量與先前研究不一致。

在先前的研究報(bào)道中也表明WRKY轉(zhuǎn)錄因子可能參與了苦痘病的發(fā)生與發(fā)展[18，22]。對(duì)蘋果Md-CAX11基因啟動(dòng)子區(qū)域進(jìn)行預(yù)測(cè)分析，發(fā)現(xiàn)基因啟動(dòng)子區(qū)存在著WRKY轉(zhuǎn)錄因子結(jié)合位點(diǎn)WBOXN-TERF3、WBOXATNPR1和WRKY71OS。因此在下一步的工作中將開展WRKY轉(zhuǎn)錄因子與MdCAX11基因啟動(dòng)子的互作分析，以期明確WRKY轉(zhuǎn)錄因子與苦痘病發(fā)生的相關(guān)性。

4結(jié)論

MdCAX11基因的鈣轉(zhuǎn)運(yùn)能力受N末端自抑制區(qū)的影響，sMdCAX11基因的過表達(dá)顯著降低植物體內(nèi)鈣含量，打破了礦質(zhì)元素比例的平衡。

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