嗜盐菌中甘氨酸甜菜碱的合成途径及其生物学功能
张英杰¹, 廖子亚^1,2, 赵百锁¹
1. 中国农业科学院研究生院, 北京 100081;
2. 重庆市辅仁中学校, 重庆 400066
收稿日期：2019-11-16；修回日期：2020-01-09；网络出版日期：2020-01-21
基金项目：国家自然科学基金（31370158, 31570110）；中国农业科学院研究生院基本科研业务费项目（610042017001）
_*通信作者：赵百锁, 男, 中国农业科学院研究生院, 研究员, 环境微生物学专业, 《微生物学通报》常务编委, 国家自然科学基金委评议专家, 中国农业微生物专业委员会委员, ISE学会会员和ISME学会会员。2006年至2012年在清华大学, University of Georgia和Washington State University从事研究工作。长期从事盐碱菌抗逆机制及其在地球循环中的作用等。共主持国家自然科学基金3项及省部级5项。参加美国国家自然科学基金、美国太空署科学研究局、美国能源部等20余项目。在国内外微生物主流期刊上发表论文70篇, 以第一及通讯作者发表 40余篇。受邀撰写了Bergey's Manual of Systematics of Archaea and Bacteria, Chapter Alkalicoccus.Published by John & Sons Press和Halophiles:Genetics and Genomes, Chapter 5.107-128, Published by Caister Academic Press英文著作, 参与《微生物肥料生产应用技术百问百答李俊等著中国农业出版社》中文著作的撰写。申请授权中国国家发明专利5项和美国国家专利1项。Tel:+86-10-82106784;E-mail:bszhao@163.com.

摘要：在嗜盐菌长期的盐适应或短期的盐胁迫过程中，甘氨酸甜菜碱（又名三甲基甘氨酸，N，N，N-trimethylglycine）发挥着极为重要的作用。甘氨酸甜菜碱在嗜盐菌中的生物合成有2种途径：胆碱氧化途径和甘氨酸甲基化途径。前者以胆碱为底物，由胆碱脱氢酶（choline dehydrogenase，BetA）和甜菜碱乙醛脱氢酶（betaine aldehyde dehydrogenase，BetB）经2次氧化生成甜菜碱；后者以甘氨酸作为底物，由甘氨酸肌醇甲基转移酶（glycine sarcosine N-methyltransferase，GSMT）和肌氨酸二甲基甘氨酸甲基转移酶（sarcosine dimethylglycine N-methyltransferase，SDMT）经3次N-甲基化生成甜菜碱。目前在JGI-IMG和EZBioCloud数据库中公布了134株嗜盐菌标准菌株的全基因组序列。其中，约56.0%的嗜盐细菌和约39.6%的嗜盐古菌拥有胆碱氧化途径所需的2个基因；约9.7%的嗜盐细菌和约0.7%的嗜盐古菌携带甲基化途径所需的2个基因。其中，8株嗜盐细菌同时拥有胆碱氧化途径和甘氨酸甲基化途径所需的全部基因。甘氨酸甜菜碱生物合成基因在典型微生物菌株或经济作物中的表达可以提高其耐盐抗逆能力，这种独特的优势已经引起科学家们强烈的兴趣，相信未来，嗜盐菌中甘氨酸甜菜碱生物合成领域内的科学理论和技术应用会有重大的突破。
关键词：嗜盐菌甘氨酸甜菜碱胆碱氧化途径甘氨酸甲基化途径
Glycine betaine: biosynthesis and biological function in halophilic microorganisms
Zhang Yingjie¹, Liao Ziya^1,2, Zhao Baisuo¹
1. Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China;
2. Chongqing Furen High School, Chongqing 400066, China
Received: 16 November 2019; Revised: 9 January 2020; Published online: 21 January 2020
_*Corresponding author: Baisuo Zhao.Tel: +86-10-82106784;E-mail:bszhao@163.com.
Foundation item: Supported by the National Natural Science Foundation of China (31370158, 31570110) and by the Graduate School of Chinese Academy of Agricultural Sciences (610042017001)

Abstract: Glycine betaine (i.e. trimethylglycine, N, N, N-trimethylglycine) plays a crucial role in halophilic microorganisms subjected to the long-term salt-stress and short-term salt-exposure. Two separate biosynthetic pathways of glycine betaine: the oxidation of choline and the methylation from glycine were found. The former is choline dehydrogenase (BetA) and betaine aldehyde dehydrogenase (BetB) using choline as substrate by two oxidization; the latter contains three reactions by N-methylation catalyzed with glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT). The genome sequences of 134 halophilic type strains were obtained online in both JGI-IMG and EZBiocloud. Approximate 56.0% of halophilic bacteria and 39.6% of halophilic archaea harbors two genes of choline oxidation. About 9.7% of halophilic bacteria and 0.7% halophilic archaea possess two genes of methylation from glycine. Among them, 8 halophilic bacteria have the genes for both choline oxidation and glycine methylation. The expression of the biosynthesis genes of glycine betaine in model microorgainsms or crops can improve their ability to resist salt torlerence. This unique feature derived from the glycine betaine has attracted scientists' strongly interest and their emergence in modern biotechnology. In the future, the scientific theory and practical application derived from of the biosynthesis of glycine betaine in halophiles must have a significant impact.
Keywords: halophilic microorganismsglycine betainecholine oxidation pathwayglycine de novo methylation pathway
在嗜盐菌长期的盐适应或短期的盐胁迫过程中，甘氨酸甜菜碱(Glycine Betaine，又称三甲基甘氨酸，N, N, N-trimethylglycine，C₅H₁₁NO₂)发挥着十分重要的作用^[1-4]。甘氨酸甜菜碱属于甘氨酸的一种衍生物(图 1)，具有3个甲基和双电荷性质且电荷在分子内分布呈中性；它分子量小(Mw=117.15)，是一种季铵型生物碱，具有亲水性和亲脂性、易溶于水，是一种极为重要的相容性溶质。在高盐条件下，该物质可以被嗜盐菌从外界环境中吸收或通过生物合成来维持胞内的高渗透压，以适应细胞外高盐或盐胁迫环境。同时，它能稳定细胞结构，保持酶、蛋白质复合物的活性和细胞膜的完整性，来抵御外部环境中盐碱、高温或冷冻条件导致的迫害^{[3, 5]}。此外，在低盐条件下，该物质也可以被嗜盐菌作为碳源或氮源利用^[6]。

图 1 甘氨酸甜菜碱的结构图 Figure 1 Structure of glycine betaine.

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迄今为止，学术界已发表多篇有影响力的有关嗜盐菌盐适应机制(“相容性溶质机制”和“内盐机制”)及其在生物技术中的应用等综述^{[1-5, 7-9]}。虽然相容性溶质的重要成员之一甘氨酸甜菜碱(以下简称为“甜菜碱”)的2种合成基因及途径的报道尚有一些，但是其合成途径在不同嗜盐菌中的分布特点及潜在应用却鲜有报道。基于目前微生物基因组数据库中已公开的嗜盐菌标准菌株的全基因组序列，本综述将对嗜盐菌(特指微生物适宜生长的盐度在0.5 mol/L以上的纯培养标准菌株)中合成甜菜碱2种途径的分布特点，嗜盐菌之间的亲缘关系及其在现代生物技术的应用，以及本领域内潜在的前沿性研究热点等方面进行述评。希望为后续的研究工作提供新思路，以激发更多****在该研究领域内的兴趣并取得丰硕的科学研究成果。
1 嗜盐菌中甜菜碱的合成途径 大多数嗜盐菌常常从外界环境摄取外源性甜菜碱来适应长期的高盐度环境和应对短期的盐胁迫^{[2, 5, 9]}。但是，若环境中缺乏甜菜碱时，一些嗜盐菌可以通过生物合成来应对极端条件，避免细胞死亡从而得以生存繁殖^{[7, 10-11]}。因此，甜菜碱的生物合成在有些嗜盐菌中成为必不可少的环节且发挥着很重要的作用。
据报道^{[2, 4, 11]}，嗜盐菌通过2种途径来合成甜菜碱：①胆碱氧化合成途径：即以胆碱为底物，利用转运蛋白运输到细胞内的胆碱首先被胆碱脱氢酶(choline dehydrogenase，BetA，betA编码)氧化为甜菜碱乙醛，然后被甜菜碱乙醛脱氢酶(betaine aldehyde dehydrogenase，BetB，betB编码)再氧化为甜菜碱(图 2-A)。采用胆碱途径合成甘氨酸甜菜碱的嗜盐细菌来源种类比较多，共有75株(图 3，表 1)。其中，代表性菌株伸长盐单胞菌Halomonas elongate DSM3043 (革兰氏阴性嗜盐细菌的模式菌株)和达坂喜盐芽孢杆菌Halobacillus dabanensis D-8^T (革兰氏阳性嗜盐细菌的模式菌株)的甜菜碱胆碱合成途径被广泛报道^[12-14]。②甘氨酸甲基化途径：即以甘氨酸为底物，S-腺苷- L-甲硫氨酸(S-adenosyl-L-methionine，AdoMet)作为甲基供体，通过3次连续的N-甲基化，从甘氨酸依次合成肌氨酸、二甲基甘氨酸和甜菜碱。催化该过程的为2个S-腺苷甲硫氨酸依赖性甲基转移酶(甘氨酸肌氨酸甲基转移酶glycine sarcosine N-methyltransferase，GSMT，gsmA编码；肌氨酸二甲基甘氨酸甲基转移酶sarcosine dimethylglycine N-methyltransferase，SDMT，sdmA编码) (图 2-B)。采用甘氨酸甲基化途径合成甘氨酸甜菜碱的嗜盐菌有14株(图 4，表 2)。深入研究的嗜盐细菌有3株，分别为嗜盐放线多孢菌Actinopolyspora halophila ATCC27976^T、盐绿需盐红螺菌Ectothiorhodospira halochloris ATCC35916^T (现为Halorhodospira halochloris A^T)、盐生隐杆藻Aphanothece halophytica strain 7418^{[11, 15-17]}。2003年，Waditee等首次分离并实验证明了嗜盐蓝细菌A. halophytica strain 7418中GSMT和SDMT参与甜菜碱合成的三个甲基化步骤^[17]。此外，嗜盐古菌葡萄牙甲烷嗜盐菌Methanohalophilus portucalensis FDF1^T合成的MpGSMT催化3步甲基化反应的第1步和第2步^[10]。MpGSMT可以分为MpGMT和MpSMT，分别催化甘氨酸甲基化为肌氨酸，肌氨酸甲基化为二甲基甘氨酸。MpSDMT催化甲基化反应的第2步和第3步，MpSDMT可以分为MpSMT和MpDMT，MpDMT催化二甲基甘氨酸甲基化为甜菜碱。研究还发现，基因gsmt和sdmt在排列上有一段重复序列，可能编码相同的酶蛋白序列^[10]。嗜盐古菌中存在甜菜碱从头合成途径是令人惊喜的发现，打破了以往仅仅以“内盐机制”为主的观点，也为研究嗜盐古菌中“相容性溶质机制”提供了新视角。最新研究发现^{[6, 18]}，伍氏醋酸杆菌Acetobacterium woodii及需盐色盐杆菌Chromohalobacter salexigens DSM 3043^T在甘氨酸甲基化合成途径中，产物甜菜碱反馈抑制作用于GSMT上，因此在甘氨酸从头合成途径中，可能发挥关键酶作用的酶为GSMT。

图 2 嗜盐菌中甘氨酸甜菜碱的2种合成途径 Figure 2 Two synthetic pathways of glycine betaine in halophilic bacteria. A: choline oxidation pathway; B: glycine methylation pathway.

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图 3 基于16S rRNA基因序列的胆碱氧化合成途径嗜盐细菌的亲缘图 Figure 3 Phylogenetic relationship based on 16S rRNA gene sequence of halophilic bacteria that synthesize glycine betaine through the choline pathway. The scale bar indicates the phylogenetic distance.

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表 1. 采用胆碱氧化途径合成甘氨酸甜菜碱的嗜盐细菌 Table 1. Halophilic bacteria that synthesize glycine betaine through choline oxidation pathway

Species	Accession number	[Na+] opt./(mol/L)	[Na+] range/(mol/L)	pH opt.	pH range
Actinopolyspora halophila DSM 43834T	AQUI01000002	2.56–3.42	1.71–5.13	ND	ND
Aestuariibacter salexigens DSM 15300T	AY207502	0.34–1.03	0.17–1.71	7.0–8.0	ND
Afifella marina DSM 2698T	FMVW01000007	0.17–0.85	0–0.68	6.9–7.1	ND
Alcanivorax borkumensis DSM 11573T	AM286690	0.51–1.71	0.17–2.14	ND	ND
Alcanivorax dieselolei DSM 16502T	CP003466	0.51–1.28	0.17–2.56	ND	ND
Alcanivorax pacificus W11-5T	AJGP01000039	0.51–0.85	0.08–2.05	ND	ND
Alkalicoccus saliphilus DSM 15402T	AJ493660	2.74	0.17–4.27	9.0	6.5–10.0
Alkalicoccus halolimnae BZ-SZ-XJ29T	KX618877	1.42–2.10	0.74–4.15	7.5	6.0–10.5
Arhodomonas aquaeolei ATCC 49307T	M26631	2.56	1.03–3.42	7.0	6.0–8.0
Bacillus aidingensis DSM 18341T	DQ504377	2.05	1.37–5.64	7.2	6.0–9.5
Bacillus atrophaeus DSM7264T	AB021181	1.20	ND	5.6–5.7	ND
Bacillus chagannorensis DSM 18086T	AM492159	1.20	0.51–3.42	8.5	5.8–10.0
Bacillus selenitireducens DSM 15326T	ABHZ01000019	0.41–1.03	0.34–3.76	8.5–10.0	ND
Bacillus urumqiensis BZ-SZ-XJ18T	KM066107	1.80	0.22–4.32	8.5–9.5	6.5–10.0
Candidatus Chlorothrix halophila	AY395567	1.71	0.85–2.05	7.8–8.4	ND
Chromohalobacter salexigens DSM 3043T	CP000285	1.28–1.71	0.15–4.27	7.5	5.0–10.0
Desulfohalobium retbaense DSM 5692T	CP001734	1.71	0–4.10	6.5–7.0	5.5–8.0
Desulfovermiculus halophilus DSM 18834T	DQ139408	1.37–1.71	0.51–3.93	7.2	6.4–8.2
Desulfovibrio bastinii DSM 16055T	AY359868	0.68	0.17–2.05	5.8–6.2	5.2–7.4
Desulfovibrio gracilis DSM 16080T	FUYC01000042	0.85–1.03	0.34–2.05	6.8–7.2	5.4–8.4
Desulfovibrio oxyclinae DSM 11498T	U33316	0.85–1.71	0.43–3.85	7.0–7.5	ND
Desulfovibrio vietnamensis DSM 10520T	X93994	0.85	0–1.71	7.5	5.0–10.0
Ectothiorhodospira haloalkaliphila ATCC 51935T	KK214998	0.85	0.43–2.56	8.5–10	ND
Enterovibrio calviensis DSM 14347T	AF118021	0.43–0.60	0.26–2.05	ND	ND
Flexistipes sinusarabici DSM 4947T	CP002858	0.51	0.51–3.08	ND	6.0–8.0
Fodinicurvata fenggangensis DSM 21160T	JMLV01000013	0.85	0.26—3.42	7.5	6.5–8.5
Gracilibacillus lacisalsi DSM 19029T	DQ664540	0.85–1.20	0.17–3.08	7.5–8.0	5.5–10
Hahella ganghwensis DSM 17046T	AQXX01000025	0.68–1.03	0.17–1.71	7.0–8.0	5.0–10.0
Halalkalibacillus halophilus DSM 18494T	AB264529	1.71–2.56	0.85–4.27	8.5–9.0	5.5–10.0
Halobacillus halophilus DSM 2266T	HE717023	0.51–0.85	0–2.56	7.8	7.0–9.0
Halobacillus kuroshimensis DSM 18393T	AB195680	1.03	0.08–4.27	7.5–8.0	5.5–10.0
Haloglycomyces albus DSM 45210T	AZUQ01000001	1.37–2.05	0.51–3.08	7.0–7.5	5.0–9.0
Halomonas anticariensis FP35T	ASTJ01000004	1.28	0.08–2.56	ND	6.0–9.0
Halomonas elongata DSM 2581T	FN869568	0.60–1.37	0–5.47	ND	5.0–9.0
Halomonas halocynthiae DSM 14573T	AJ417388	0.51–1.03	0.08–2.56	7.5–8.5	5.0–11.0
Halomonas halodenitrificans DSM 735T	L04942	0.30–2.0	0–5.5	ND	ND
Halomonas ilicicola DSM 19980T	EU218533	1.71	0.34–3.0	6.5	6.0–9.0
Halomonas jeotgali HwaT	AMQY01000015	1.71	0.85–4.27	7.0–8.0	5.0–10.0
Halomonas lutea DSM 23508T	ARKK01000003	0.85–1.71	0.17–3.42	7.5	5.0–9.0
Halomonas urumqiensis BZ-SZ-XJ27T	KM066108	1.42	0.22–4.32	8.0–8.5	6.0–10.0
Halomonas zhanjiangensis DSM 21076T	FJ429198	0.51–0.85	0.17–3.42	7.5	6.0–10.5
Halonatronum saccharophilum DSM 13868T	AZYG01000049	1.20–2.05	0.51–2.91	8.0–8.5	7.7–10.3
Idiomarina loihiensis L2TRT	AE017340	1.28–1.71	ND	ND	ND
Kushneria aurantia A10T	AM941746	1.71	0.85–3.0	7.0–8.0	5.5–8.5
Kushneria indalinina CG2.1T	AJ427627	1.28–1.71	0.51–4.27	7.2	5.0–9.0
Lentibacillus juripiscarius JCM 12147T	AB127980	1.71	0.51–5.13	7.0	5.0–9.0
Limimaricola hongkongensis DSM 17492T	APGJ01000007	ND	0.34–2.39	ND	5.0–10.0
Litorimicrobium taeanense DSM 22007T	jgi.1055387	0.34–0.68	0.17–1.03	6.5–7.5	6.0–8.0
Marinobacter algicola DG893T	ABCP01000031	0.51–1.03	0.17–2.05	7.5	5.0–10.0
Marinobacter daepoensis DSM 16072T	AY517633	0.34–1.03	0–3.08	7.0–8.0	5.5–ND
Marinobacter hydrocarbonoclasticus ATCC 49840T	FO203363	0.60	0.08–3.5	7.0–7.5	6.0–9.5
Marinobacterium halophilum DSM 17586T	AY563030	ND	0.51–2.05	ND	5.3–9.3
Marinobacterium lutimaris DSM 22012T	jgi.1107934	0.34–0.85	0.17–1.71	6.5–7.5	6.0–8.0
Marinomonas ushuaiensis DSM 15871T	AJ627909	1.03	0.17–ND	8.0	7.0–12.5
Methylophaga aminisulfidivorans KCTC 12909T	AFIG01000001	0.51	0.26–1.54	6.8–7.0	6.0–8.0
Nocardiopsis chromatogenes YIM 90109T	AY619715	0.85–1.37	0–3.08	7.2	6.0–9.0
Nocardiopsis gilva YIM 90087T	AY619712	0.85–1.37	0–3.08	7.2	6.0–9.0
Nocardiopsis salina YIM 90010T	AY373031	1.71	0.51–3.42	7.2	6.0–9.0
Oceanicella actignis DSM 22673T	JQ864435	0.34–0.85	0.17–1.54	7.5–8.0	5.5–9.5
Pontibacillus marinus BH030004T	AVPF01000156	0.34–0.85	0.17–1.54	7.0–7.5	6.0–9.0
Rubidibacter lacunae KORDI 51-2T	ASSJ01000063	0.85	0.34–1.20	ND	ND
Ruegeria lacuscaerulensis ITI-1157T	ACNX01000031	0.60	0–1.20	7.5	ND
Saccharomonospora halophila 8T	AICX01000084	1.71	1.71–5.13	ND	ND
Saccharomonospora paurometabolica YIM 90007T	AF540959	1.71	ND	ND	ND
Salinibacter ruber DSM 13855T	CP000159	2.56–5.13	2.56–ND	6.5–8.0	6.0–8.5
Salinicoccus albus DSM 19776T	EF177692	1.71	0.17–5.13	8.5	6.0–10.0
Salinicoccus kunmingensis DSM 17847T	DQ837380	1.36–1.71	0.08–4.27	8.0	6.0–10.0
Salinimonas chungwhensis DSM 16280T	AY553295	0.34–0.85	0–2.56	7.0–8.0	ND
Salipiger mucosus DSM 16094T	ARRM01000007	1.54–1.71	0.08–3.42	ND	6.0–10.0
Salsuginibacillus kocurii DSM 18087T	AM492160	1.71	0.51–3.42	8.5	5.8–10.0
Sedimentitalea nanhaiensis DSM 24252T	AXBG01000021	0.17–0.68	0.10–1.03	7.0–8.5	6.0–9.3
Streptomyces zinciresistens K42T	AGBF01000432	0.68	0.68–1.20	7.5	5.0–11.0
Thalassospira profundimaris WP0211T	AY186195	0.51–0.68	0.34–1.37	ND	ND
Thalassospira xiamenensis DSM 17429T	CP004388	0.34–0.68	0.08–1.71	ND	ND
Thioalkalivibrio paradoxus ARh 1T	AF151432	0.50–1.00	0.30–4.30	10.2	ND
ND: Data not shown in the published paper.

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图 4 基于16S rRNA基因序列的甘氨酸甲基化合成途径嗜盐菌的亲缘图 Figure 4 Phylogenetic relationship based on 16S rRNA gene sequence of halophiles that synthesize glycine betaine through glycine methylation pathway. The scale bar indicates the phylogenetic distance. Numbers at the branch nodes are bootstrap values for 1000 replicates.

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表 2. 采用甘氨酸甲基化途径合成甘氨酸甜菜碱的嗜盐菌 Table 2. Halophilic bacteria that synthesize glycine betaine through glycine methylation pathway

Species	Accession number	[Na+] opt./(mol/L)	[Na+] range/(mol/L)	pH opt.	pH range
Actinopolyspora halophila DSM 43834T	AQUI01000002	2.56–3.42	1.71–5.13	ND	ND
Aphanothece halophytica ATCC 43922T	AJ000721	1.00–4.00	0–5.0	ND	ND
Arhodomonas aquaeolei DSM 8974	M26631	2.56	1.03–3.42	6.5–7.5	6.0–8.0
Candidatus Chlorothrix halophile	AY395567	1.71	0.85–2.05	7.8–8.4	ND
Desulfonatronospira thiodismutans ASO3-1T	ACJN01000005	1.71	0–3.08	9.0	7.0–10.0
Ectothiorhodospira haloalkaliphila ATCC 51935T	KK214998	0.85–1.03	0.43–2.56	8.5–10.0	ND
Fodinicurvata fenggangensis DSM 21160T	JMLV01000013	0.85	0.26–3.42	7.5	6.5–8.5
Halorhodospira halochloris AT	AP017372	2.40–4.62	1.54–5.13	8.1–9.1	ND
Halorhodospira halophila SL1T	CP000544	1.88–3.76	1.54–5.13	7.4–7.9	ND
Methanohalophilus portucalensis FDF1T	JWTK01000010	0.51–2.56	ND	6.5–7.5	ND
Natranaerobius thermophilus JW/NM-WN-LFT	CP001034	3.90	3.10–4.90	9.5	8.3–10.6
Rubidibacter lacunae KORDI 51-2T	ASSJ01000063	0.85	0.34–1.20	ND	ND
Saccharomonospora paurometabolica YIM 90007T	AF540959	1.71	ND	ND	ND
Thioalkalivibrio paradoxus ARh 1T	AF151432	0.50–1.00	0.30–4.30	10.2	ND
ND: Data not shown in the published paper.

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2 甜菜碱来源于多种类型的嗜盐菌 截止笔者撰写时，从JGI-IMG (https://img.jgi.doe.gov/cgi-bin/mer/main.cgi)和EZBioCloud (https://www.ezbiocloud.net)数据库中共获得了134株含有甜菜碱生物合成基因的嗜盐菌标准菌株。其中，75株嗜盐细菌(占总数~56.0%，表 1)和53株嗜盐古菌(~39.6%，表 3)含有胆碱氧化途径所需的2个基因；13株嗜盐细菌(~9.7%)和1株嗜盐古菌(~0.7%)拥有甘氨酸甲基化途径所需的2个基因(表 2)。令人兴奋的是，我们发现有8株嗜盐细菌同时携带甜菜碱2种生物合成途径所需的基因(表 4)，说明他们的盐适应调控机制可能存在独特性和复杂性，此类科学问题在以往的报道中还没有提及。因此，共存的2种甜菜碱合成途径在这8株嗜盐细菌中究竟是如何发挥作用，是否存在优先合成机制等还需要更深入的研究。本课题组正在从生理生化、分子遗传和组学工具(基因组、转录组和蛋白组)等方面进行全面的、系统的和深入的研究，期望不久的将来揭开2种甜菜碱生物合成共存途径的科学面纱。
表 3. 采用胆碱氧化途径合成甘氨酸甜菜碱的嗜盐古菌 Table 3. Halophilic archaea that synthesize glycine betaine through choline oxidation pathway

Species	Accession number	[Na+] opt./(mol/L)	[Na+] range/(mol/L)	pH opt.	pH range
Haloferax alexandrinus JCM 10717T	AB037474	4.27	1.71–5.30	7.2	5.5–7.5
Haloferax denitrificans S1T	AOLP01000013	2.00–3.00	1.50–4.50	6.7	6.0–8.0
Haloferax elongans ATCC BAA-1513T	DQ860977	2.60–3.40	1.70–5.10	7.4	7.0–9.0
Haloferax gibbonsii ATCC 33959T	AOLJ01000012	2.56–4.27	1.71–5.30	6.5–7.0	5.5–8.0
Haloferax larsenii JCM 13917T	AOLI01000021	2.20–3.40	1.00–4.80	6.5–7.0	6.0–8.5
Haloferax lucentense DSM 14919T	AOLH01000027	4.27	1.71–5.13	7.5	5.0–9.0
Haloferax mediterranei R-4T	CP001868	2.90	2.05–4.27	6.5	ND
Haloferax mucosum PA12T	AOLN01000016	2.60–3.40	1.70–5.10	7.4	6.0–10.0
Haloferax prahovense TL6T	AB258305	3.50	2.50–5.20	7.0–7.5	6.0–8.5
Haloferax sulfurifontis M6T	AOLM01000015	ND	1.03–ND	6.4–6.8	4.5–9.0
Haloferax volcanii DS2T	CP001956	1.70–2.50	ND	ND	ND
Halobaculum gomorrense DSM 9297T	L37444	1.50–2.50	1.00–ND	6.0–7.0	5.5–8.0
Halogeometricum borinquense DSM 11551T	ABTX01000001	3.42–4.27	1.37–ND	ND	ND
Halogeometricum pallidum JCM 14848T	AOIV01000004	3.70	1.70–4.30	6.5	5.0–8.5
Haloplanus natans DSM 17983T	KE386573	3.00	2.60–4.30	7.0	6.5–8.0
Halorubrum aidingense JCM 13560T	DQ355813	2.60	1.70–4.30	7.5	7.0–9.0
Halorubrum arcis AJ201T	DQ355793	3.40–3.90	2.20–5.20	7.5	6.0–8.5
Halorubrum californiense SF3-213T	EF139654	3.50–4.50	2.50–5.00	7.3	6.8–8.5
Halorubrum coriense Ch2T	L00922	ND	1.50–5.30	ND	ND
Halorubrum ezzemoulense DSM 17463T	DQ118426	3.42	2.56–4.27	7.0–7.5	6.5–9.0
Halorubrum kocurii JCM 14978T	AOJH01000060	3.42	2.50–ND	7.5	6.0–9.
Halorubrum lipolyticum DSM 21995T	DQ355814	2.60	1.70–4.80	7.5	7.0–9.0
Halorubrum litoreum JCM 13561T	AOJF01000057	3.42	2.00–5.10	7.0–7.5	6.5–9.0
Halorubrum saccharovorum M6T	U17364	3.50–4.50	1.50–5.20	ND	ND
Halorubrum distributum JCM 9100T	D63572	4.27	1.50–5.30	ND	ND
Halorubrum sodomense DSM 3755T	jgi.1055354	1.70–2.50	0.50–4.30	ND	ND
Halorubrum tebenquichense DSM 14210T	AOJD01000002	ND	2.50–5.20	ND	7.0–10.0
Halorubrum terrestre JCM 10247T	AB090169	4.27	2.56–5.13	7.5	5.0–9.0
Halopiger xanaduensis SH-6T	CP002839	4.30	2.50–5.00	7.5–8.0	6.0–11.0
Halobiforma lacisalsi AJ5T	CP019285	2.60–4.30	1.70–ND	7.5	6.5–9.0
Haloterrigena limicola JCM 13563T	DQ367241	3.10	1.70–5.10	7.0	6.5–9.0
Haloterrigena saccharevitans AB14T	LWLN01000002	3.00–3.40	1.70–ND	7.5	6.5–8.5
Haloterrigena salina JCM 13891T	AM942763	3.40	2.50–5.00	7.0–8.0	6.0–9.0
Halovivax asiaticus JCM 14624T	AOIQ01000016	3.40	2.50–4.30	7.0–7.5	6.0–9.0
Halovivax ruber XH-70T	CP003050	3.40	2.50–4.30	7.0–7.5	6.0–9.0
Natrinema altunense JCM 12890T	JNCS01000019	3.00–4.30	1.70–ND	7.0–7.7	6.0–8.0
Natrinema gari JCM 14663T	AB289741	2.60–3.40	1.70–5.1	6.0–6.5	5.5–8.5
Natrinema pallidum DSM 3751T	AJ002949	3.40–4.30	1.70–ND	7.2–7.6	6.0–8.4
Natrinema pellirubrum DSM 15462T	AGIN01000009	3.40–4.30	2.00–ND	7.2–7.8	6.0–8.6
Natrinema versiforme JCM 10478T	AB023426	3.40–4.30	1.50–ND	6.5–7.0	6.0–8.0
Natronococcus jeotgali DSM 18795T	EF077631	3.93–4.27	1.28–5.13	7.5	7.0–9.5
Natronorubrum tibetense GA33T	KB913017	3.40	2.00–5.10	9.0	8.5–11.0
Natronococcus occultus SP4T	CP003929	2.80	1.40–5.20	9.5	8.5–11.0
Haladaptatus paucihalophilus DX253T	AQXI01000001	2.60–3.10	0.80–5.10	6.0–6.5	5.0–7.5
Halalkalicoccus jeotgali B3T	CP002062	2.56	1.71–5.13	7.0	6.5–9.0
Haloarcula amylolytica JCM 13557T	DQ826512	2.90–3.20	2.00–5.10	7.0–7.5	6.5–9.0
Haloarcula argentinensis DSM 12282T	EF645681	2.50–3.00	2.00–4.50	ND	ND
Haloarcula japonica DSM 6131T	AB355986	3.42	2.56–5.13	7.0–7.5	6.0–8.0
Halococcus saccharolyticus DSM 5350T	AOMD01000030	4.27	2.56–5.3	7.2	6.0–8.0
Halococcus salifodinae DSM 8989T	AOME01000075	3.42–4.27	2.56–ND	ND	6.8–9.5
Halosimplex carlsbadense JCM 11222T	HQ263562	4.27	3.42–5.13	ND	7.0–8.0
Natronomonas moolapensis DSM 18674T	HF582854	3.10–3.42	2.39–6.15	7.0–7.5	5.5–8.5
Salinigranum rubrum GX10T	GU951431	3.10	1.40–4.80	7.0	5.0–9.0
ND: Data not shown in the published paper.

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表 4. 同时含有甘氨酸甲基化途径和胆碱氧化途径的嗜盐细菌 Table 4. Halophilic bacteria that contain both glycine methylation pathway and choline oxidation pathway

Species	Accession number	[Na+] opt./(mol/L)	[Na+] range/(mol/L)	pH opt.	pH range
Actinopolyspora halophila DSM 43834T	AQUI01000002	2.56–3.42	1.71–5.13	ND	ND
Saccharomonospora paurometabolica YIM 90007T	AF540959	1.71	ND	ND	ND
Arhodomonas aquaeolei DSM 8974	M26631	2.56	1.03–3.42	6.5–7.5	6.0–8.0
Ectothiorhodospira haloalkaliphila ATCC 51935T	KK214998	0.85–1.03	0.43–2.56	8.5–10	ND
Fodinicurvata fenggangensis DSM 21160T	JMLV01000013	0.85	0.26–3.42	7.5	6.5–8.5
Thioalkalivibrio paradoxus ARh 1T	AF151432	0.50–1.00	0.30–4.30	10.2	ND
Candidatus Chlorothrix halophile	AY395567	1.71	0.85–2.05	7.8–8.4	ND
Rubidibacter lacunae KORDI 51-2T	ASSJ01000063	0.85	0.34–1.20	ND	ND
ND: Data not shown in the published paper.

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采用胆碱氧化途径合成甜菜碱的嗜盐细菌标准菌株在门分类水平上，依次为变形菌门(Proteobacteria，占总数~61.3%)、厚壁菌门(Firmicutes，~22.7%)、放线菌门(Actinobacteria，~10.7%)，其余则不足2%，分布在脱铁杆菌门(Deferribacteres)、绿弯菌门(Chloroflexi)、蓝细菌门(Cyanobacteria)和Rhodothermaeota (原属于Bacteroidetes^[19])。在纲的水平上，依次为γ-变形菌纲(γ-Proteobacteria，~40.0%)、α-变形菌纲(α-Proteobacteria，~13.3%)、芽孢杆菌纲(Bacilli，~20.0%)、δ-变形菌纲(δ-Proteobacteria，~8.0%)和放线菌纲(Actinobacteria，~10.0%)。此外，梭菌纲(Clostridia)、脱铁杆菌纲(Deferribacteres)、绿球菌纲(Chroobacteria)和绿弯菌纲(Chloroflexia)等所占比例都小于2% (图 3和表 1)。其中，本课题组测序分析了变形菌门中的Halomonas urumqiensis BZ-SZ-XJ27^T，以及厚壁菌门中的Bacillus urumqiensis BZ-SZ-XJ18^T和Alkalicoccus saliphilus DSM 15402^T全基因组序列^[20-23]。系统在嗜盐细菌适应极端环境中发育分析表明，这些嗜盐细菌在亲缘关系上相距甚远(图 3)，但是它们都编码胆碱氧化途径所需的胆碱脱氢酶(BetA)和甜菜碱乙醛脱氢酶(BetB)，由此可见胆碱氧化途径以及所合成的甜菜碱的作用举足轻重。
而采用胆碱氧化途径合成甜菜碱的嗜盐古菌都属于广古菌门(Euryarchaeota)中的盐杆菌纲(Halobacteria) (图 5和表 3)。在科的水平上，仅有1株菌属于Halorubraceae，其余菌株依次被归类为盐富饶菌科(Haloferacaceae，占总数~52.8%)、盐细菌科(Halobacteriaceae，~17.0%)和钠白菌科(Natrialbaceae，~28.3%)。合成甜菜碱的嗜盐古菌多数被归类于盐富饶菌科，这类菌的嗜盐浓度一般都高于嗜盐细菌，对Na⁺的适应浓度普遍大于4.0 mol/L。一般而言，嗜盐古菌采用“内盐机制”来适应高盐环境^[24-25]。但是，我们也发现有多种类型的嗜盐古菌携带甜菜碱胆碱氧化合成所需基因，暗示着一些嗜盐古菌也有可能应用“相容性溶质机制”来抵御外界的高盐条件。以上假说还需要更多的理论及实验数据来验证。究竟哪一种机制为主还是二者相辅相成，需要进一步阐明。

图 5 基于16S rRNA基因序列的胆碱氧化合成途径嗜盐古菌的亲缘图 Figure 5 Phylogenetic relationship based on 16S rRNA gene sequence of halophilic archaea that synthesize glycine betaine through choline pathway. The scale bar indicates the phylogenetic distance.

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相对于胆碱氧化途径，嗜盐菌中的甘氨酸甲基化途径的报道还比较少^{[10-11, 15-17, 26-27]}，数据库分析表明，仅有14株嗜盐菌含有甘氨酸甲基化途径所需的基因(图 4和表 2)。从门分类水平上，依次为变形菌门(占总数~50.0%)、放线菌门(~14.3%)、蓝细菌门(~14.3%)，而厚壁菌门和绿弯菌门数量上较少，均占~7%。在纲分类水平上，多数属于γ-变形菌纲(~35.7%)和放线菌纲(~14.3%)，一部分属于α-变形菌纲(~11.7%)。而δ-变形菌纲、绿弯菌纲、梭菌纲和绿球菌纲所占数量较少(~7.1%)。梭菌纲中嗜热盐碱厌氧菌Natranaerobius thermophilus JWNM-WN-LF^T是一株厌氧嗜盐、嗜碱和嗜热的多嗜极细菌，本研究组采用蛋白组学iTRAQ技术、RNA-Seq技术和数字PCR技术，在不同极端条件下检测该菌的抗逆特性，发现了甜菜碱甲基化途径相关基因的表达。实验表明，GSMT和SDMT从低盐碱热条件下(3.0 mol/L Na⁺、pH 8.8和42 ℃)到高盐碱热条件下(4.0 mol/L Na⁺、pH 9.8和52 ℃)，GSMT的基因上调1.75倍，SDMT的基因上调2.02倍(数据未发表)。此外，仅发现1株嗜盐古菌(葡萄牙甲烷嗜盐菌，Methanohalophilus portucalensis FDF1^T)采用了甘氨酸甲基化途径合成甜菜碱^[10]。值得关注的是，采用甘氨酸甲基化途径的嗜盐细菌和嗜盐古菌都编码合成甘氨酸肌醇甲基转移酶和肌氨酸二甲基甘氨酸甲基转移酶，但两种酶在嗜盐细菌和嗜盐古菌中结构和功能是否存在差异，还需要后续的实验工作来揭示其可能存在的深刻含义。
此外，有8株嗜盐细菌同时存在以胆碱或甘氨酸为底物的两种生物合成途径(表 4)，包括变形菌：水油非红单胞菌Arhodomonas aquaeolei DSM 8974^T、喜盐碱外硫红螺菌Ectothiorhodospira haloalkaliphila ATCC 51935^T、Fodinicurvata fenggangensis DSM 21160^T和Thioalkalivibrio paradoxus ARh 1^T，放线菌：嗜盐放线多孢菌Actinopolyspora halophila DSM 43834^T、寡食糖单孢菌Saccharomonospora paurometabolica YIM 90007^T，绿弯菌：Candidatus Chlorothrix halophile^T，和蓝细菌Rubidibacter lacunae KORDI 51-2^T。8株嗜盐细菌对于Na⁺的最适浓度范围为0.50–2.56 mol/L，生长浓度范围为0–4.30 mol/L。这些嗜盐菌存在两种合成途径，但两种合成途径如何相互协调发挥作用，是否存在某种诱导因素使嗜盐菌仅选择其中一种机制合成甜菜碱，这是目前相关领域的研究中没有提及的，也是值得相关****进行深入研究的很好的切入点。值得关注的是，采用甘氨酸从头合成甜菜碱是相对较为耗能的策略机制，因为甲基化反应是自然界中高耗能反应之一，每转移一个S-腺苷-L-甲硫氨酸供体需要细胞消耗12个ATP^[15]。因此甘氨酸甲基化从头合成途径在自然界中较为罕见，但仍有部分嗜盐菌采用此种途径，其中的生物学功能还需要进一步探索。
3 甜菜碱在盐适应及盐胁迫中起重要的作用 嗜盐菌中甜菜碱的生物学功能被报道了一些^{[2, 4, 9, 11, 28-29]}，但对采用不同合成途径的嗜盐菌中甜菜碱基因的作用是否存在差异鲜有报道。由于嗜盐菌不是分类学中的专有名词，不同门属之间的嗜盐菌的生理特性、携带的基因结构及其功能上存在着较大的差异。因此本文选取胆碱途径和甘氨酸途径中较为代表性的菌株进行探讨，希望对甜菜碱的盐适应机理进行系统的阐述。
作为革兰氏阴性的中度嗜盐细菌的模式菌株，伸长盐单胞菌(Halomonas elongata)中胆碱合成途径已被深入研究^{[12-13, 30-31]}。研究表明，在2.0 mol/L NaCl高盐条件下，H. elongata DSM3043细胞吸收胆碱的速率比0.5 mol/L NaCl低盐条件下快4倍。此外，在2.0 mol/L NaCl条件下，该菌株从环境中吸收胆碱合成甜菜碱的菌株传代数是合成途径受阻菌株的2倍。作为革兰氏阳性中度嗜盐细菌的模式菌株，达坂喜盐芽孢杆菌(Halobacillus dabanensis D-8^T)在1.0 mol/L和2.0 mol/L NaCl条件下，培养基中添加胆碱的OD₆₀₀值比未处理的菌株高6倍^[14]。胆碱虽然不是相容性渗透保护物质，但对于能进行生物合成甜菜碱的嗜盐菌十分重要。嗜盐古菌的盐适应机理已经得到了广泛的研究，普遍被认可的嗜盐机理为“内盐机制”，而盐杆菌纲作为研究较为透彻的嗜盐古菌，其盐适应机制已经得到了广泛的研究，早在1970s就有报道嗜盐古菌的“内盐机制”^[32-33]，即在高盐条件下嗜盐古菌通过吸收钾离子来增加细胞内的渗透压，以抵御细胞外盐环境或响应盐冲击所致的胁迫环境^{[24-25, 34]}。但是，迄今为止尚未发现关于嗜盐古菌中存在胆碱氧化途径合成甜菜碱的报道，对于嗜盐古菌中甜菜碱的报道仅仅涉及转运部分，尚无报道涉及嗜盐古菌采用胆碱氧化途径合成甜菜碱^{[2, 29, 34]}。此外，尚无本文所述的53株携带胆碱氧化途径相关基因的嗜盐古菌的报道，那么在这53株古菌中究竟还是以一种机制为主还是两者相辅相成，这将成为后续研究的突破点和创新点。
对从甘氨酸合成甜菜碱的嗜盐菌而言，甜菜碱是否也有明显促进其在高盐环境中生长的效果呢？嗜盐放线多孢菌A. halophila ATCC27976^T和盐绿需盐红螺菌E. halochloris ATCC35916^T (现为Halorhodospira halochloris A^T)是目前研究较为深入的采用甘氨酸甲基化途径的嗜盐细菌，该研究证实了两株嗜盐菌均可以在高盐环境中积累甜菜碱，4.0 mol/L NaCl环境中细胞内甜菜碱比2.0 mol/L NaCl环境中高2倍，并且已经完成相关合成蛋白的功能测定，表明该菌株在高盐刺激下甜菜碱合成蛋白发挥作用，促进甜菜碱的合成^{[11, 15-16]}。厚壁菌门中嗜热盐碱厌氧菌Natranaerobius thermophilus JWNM-WN-LF^T是一株厌氧、嗜盐、嗜碱、嗜热细菌，最佳生长条件为3.9 mol/L Na⁺ (23% NaCl，W/V)，pH 9.5，温度53 ℃，其独特的生存条件表明其细胞内必定存在不同于常见中度嗜盐菌的机制特征。据报道^[35-38]，N. thermophilus中携带甜菜碱从头合成途径相关基因，本研究组对N. thermophilus不同嗜极条件下胞内甜菜碱进行了分析(数据未发表)，结果表明N. thermophilus细胞内甜菜碱浓度随着盐浓度的增加而升高，且浓度增长幅度较大，如Na⁺浓度为4.5 mol/L (相当于约27% NaCl)时，甜菜碱浓度是Na⁺浓度为3.3 mol/L时(约19% NaCl)的3倍。对于同属于硬壁菌门的革兰氏阳性中度嗜盐菌盐反硝化枝芽孢杆菌Virgibacillus halodenitrificans PDB-F2来说，甜菜碱显著促进了菌株在6%–15% NaCl中的生长速度，15% NaCl中尤为明显，增长速度是对照组的1.28倍。这些结果表明甜菜碱对嗜盐菌适应盐环境及响应高盐胁迫冲击来说至关重要^[39]。对于生存时间较久的嗜盐古菌来说，其“相容性溶质机制”还未得到明确的认识和较为深入的研究。葡萄牙甲烷嗜盐菌Methanohalophilus portucalensis PDF1^T是目前研究最为彻底的采用甘氨酸途径合成甜菜碱的嗜盐古菌，该研究除了证明M. portucalensis在高盐环境中能合成甜菜碱外，若将相关蛋白基因转入到缺陷型菌株E.coli MKH13中，在盐环境下携带重组蛋白的菌株比缺陷型菌株在生长时间上明显加快(0.8 mol/L NaCl下提前约10 h)^[10]。
甜菜碱的重要作用不仅仅局限于盐环境，嗜热盐碱厌氧菌中甜菜碱相关合成基因的发现，我们推测该基因表达的蛋白可能存在耐受多种极端环境的特性。甜菜碱除了有作为渗透保护剂的作用外，还可作为酶功能的有效稳定剂，提供对盐度、高温、冻融处理甚至干燥等胁迫的抵抗作用^{[2, 29, 40]}。在低渗条件下，甜菜碱可以降解为二甲基甘氨酸、肌氨酸和甘氨酸等^[6]，降低细胞内渗透压并作为碳源或氮源等能源物质供应细胞生长需求。
4 甜菜碱合成基因在生物技术中的应用 虽然甜菜碱合成基因的功能鉴定方面的科学报道较多，但是其在现代生物技术应用领域的报道还较少。以下就其在基因工程菌的构建和转基因农作物中的应用作一些介绍。
(1) 胆碱途径合成基因的应用。Cánovas等(2000)将伸长盐单胞菌H. elongate DSM3043的betIBA基因导入缺陷型大肠杆菌MKH13菌株中，该菌株在0.5 mol/L盐度下，能够合成甜菜碱。Gu等(2008)将达坂喜盐芽孢杆菌H.dabanensis D-8^T中的gbsAB基因在大肠杆菌PD141中进行表达，在含有1 mmol/L胆碱的0.7 mol/L盐度下培养，能够检测到甜菜碱。Prasad等将嗜盐节杆菌Arthrobacter pascens中的胆碱氧化酶基因(codeA)转入荠菜中，发现可以促进种子萌发和植株生长。在0.2 mol/L Na⁺环境中，野生型植物无种子萌发，而携带codeA基因的植株种子萌发率可达40%^[41]。Mohanty等将codeA基因转入水稻中，50%的转基因植株在盐胁迫条件下存活并有种子生成^[42]；Su等将带有胁迫诱导启动子的cox基因导入水稻中，发现其甜菜碱的积累是组成型启动子植株2倍^[43]。此外，在拟南芥、玉米和番茄中，发现甜菜碱基因的异源表达对植物的生殖器官也有保护作用^[44]。这可能是由于甜菜碱从植物其他组织(如叶子)中转移流入到生殖器官中，从而在生殖器官内积聚了相对高浓度的甜菜碱，最终对其起到了保护作用。这说明甘氨酸甜菜碱合成基因的异源表达可以保护或增强植物多个组织器官的功能。
(2) 甘氨酸途径合成基因的应用。迄今为止，国内外已有多篇报道涉及将甘氨酸甜菜碱途径合成基因导入植物中^[45-48]。目前，盐生隐杆藻A. halophytica中的甘氨酸甜菜碱从头合成基因的异源表达是转基因领域研究的重点。将盐隐生杆藻中甜菜碱合成基因ApGSMT及ApDMT转入鱼腥藻Anabaena sp. PCC7120中，在0.12 mol/L NaCl条件下，转基因鱼腥藻的生长速率是野生型的2倍^[48-49]。将盐隐生杆藻的甜菜碱合成基因ApGSMT和ApDMT转入水稻中，转基因植株对盐胁迫的抗性高于野生型植株，在0.15 mol/L NaCl下，转基因植株表达的甜菜碱浓度为普通水稻的4–6倍。通过检测叶片中叶绿素的含量，从而确定盐胁迫对于植株的影响，在0.2 mol/L NaCl环境中，转基因植株叶片中叶绿素含量是野生型的2.6倍，在0.4 mol/L NaCl下，高达6倍^[46]。最近，Song等将盐隐生杆菌中ApGSMT2g和ApDMT2g(编码甜菜碱合成酶，此报道采用此名称)转入棉花中，结果表明转基因植株在0.15–0.20 mol/L NaCl条件下甜菜碱获得率是野生型的2倍^[47]。此外，将M. portucalensis FDF1^T中的甜菜碱合成基因Mpgsmt-sdmt导入大肠杆菌MKH13中，在0.7–0.8 mol/L盐条件下，携带合成基因的大肠杆菌共表达MpGSMT-SDMT的滞后时间缩短，对外界环境的盐适应能力增强^[10]。若将MpGSMT和MpDMT基因转入到拟南芥中，则转基因植株在0.15–0.30 mol/L NaCl环境中相较于对照组长势较好，叶片数量及根长度均约为对照组2倍^[45]。以上甘氨酸甜菜碱合成基因的拓展研究有利于耐盐基因工程菌的构建，将甜菜碱合成基因导入某些不耐盐工程菌株，可使菌株在高渗环境中发挥其生理功能，更好地应用于工农业生产实践。细胞内生物合成的甜菜碱在植物抗逆性中可发挥重要的作用，对于在盐碱环境中难以生存的经济作物，可以将甜菜碱合成基因导入植株内，从而合成甜菜碱，以增强植株抵抗盐胁迫的能力，有益于植株的生长及适应。
5 展望 甜菜碱作为一种很重要的相容性溶质，在嗜盐菌长期盐适应及短期盐胁迫过程中发挥关键性的作用。甜菜碱的生物合成有胆碱氧化途径和甘氨酸甲基化途径。目前较多的研究集中于单一合成途径，但我们发现还有一部分嗜盐细菌拥有2种合成途径，这一有趣的发现引发了我们的思考：这两种合成途径在高盐环境下是如何调节的，是否存在某一盐浓度阈值来触发不同的生物合成机制，又或是两种途径在盐环境中相互影响？这些问题有待解决。现在对于能生物合成甜菜碱的嗜盐菌(不同类别)之间的亲缘关系还缺乏系统的认识和比较，甜菜碱合成基因在各类细胞中的功能还有待深入研究。甜菜碱和其他相容性溶质相比有什么优势，嗜盐菌更偏向于利用哪种相容性溶质作为渗透保护剂也值得大家的深入思考。
由于嗜盐菌的盐适应机制是非常复杂的过程，有时包括几种相容性溶质同时发挥作用，这几种相容性溶质如何发挥作用以及它们之间存在的相互影响及作用，也是以后需要深入研究的重点。对于采用其他相容性溶质作为渗透调节剂的嗜盐菌，甜菜碱是否会对其产生渗透保护作用，又会对其本身合成的相容性溶质产生什么影响，目前此方面鲜有报道。在构建基因工程菌株方面，应用甜菜碱合成基因在现代食品工业、发酵工业、污水处理和石油污染生物修复中发挥作用，开发转基因植物以抵抗盐碱地环境是今后研究的重点内容。我国盐碱地的改良和耐盐碱农作物的开发也是改善盐碱地问题、提高农业水平的关键。综上，希望本综述提供的一些研究结果、观点以及思路能为该领域的深入研究尽一点绵薄之力，以期甜菜碱生物合成基因功能等方面的科学研究成果更加丰硕。

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