生物鐘在蛋雞排卵-產蛋過程中的調控作用

王曉鵑，劉磊，焦洪超，趙景鵬，林海

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生物鐘在蛋雞排卵-產蛋過程中的調控作用

王曉鵑，劉磊，焦洪超，趙景鵬，林海

（山東農業大學動物科技學院/山東省動物生物工程與疾病防治重點實驗室，山東泰安 271018）

生物體內源性的晝夜節律使其能夠預測周邊環境周期性的變化，使機體的內在代謝和周邊環境保持一致。在禽類卵泡的成熟、排卵和蛋的形成過程中，不同生理進程在時間上的吻合顯示了機體自身以及機體與環境之間的協調統一。動物對營養物質的攝入、內分泌激素的生成、能量代謝等一系列的行為和生理過程都有生物鐘參與調控。文章從光照和營養兩種因素入手，綜述了生物鐘在神經內分泌、能量攝入和能量代謝中的調控作用，揭示了蛋雞的排卵和產蛋機制。1.光信號通過調控生物鐘影響下丘腦-垂體-性腺軸（HPG軸），從而調控機體的繁殖活動。在光信號刺激下，位于禽類視交叉上核（SCN）和松果體的中樞生物鐘作用于下丘腦，使下丘腦定時性釋放促性腺激素釋放激素（GnRH）和促性腺激素抑制激素（GnIH），GnRH和GnIH繼而作用于垂體調節釋放促性腺激素-促黃體生成素（LH）和促卵泡激素（FSH），卵巢中存在的外周生物鐘接受中樞的同步化信號來維持生物節律，促使禽類的卵泡成熟和定時排卵；2.除了受到HPG的神經內分泌調控之外，蛋雞的排卵-產蛋過程還受到機體能量代謝的影響。中樞和外周的生物鐘基因能夠調控食欲調節系統，從而影響能量攝入；生物鐘能夠通過調控代謝過程中重要限速酶的表達、整合核受體和營養信號蛋白、調節代謝感受器和代謝物、影響腸道微生物等途徑來調節能量代謝，影響卵黃前體物質的合成、轉運和沉積；禽類松果體分泌的褪黑素可通過介導降鈣素、甲狀旁腺素（PTH）及雌激素分泌，節律性地調節體內鈣代謝，影響蛋殼的形成。能量攝入的時間和行為、機體能量代謝和能量狀態也可以通過腺苷酸活化蛋白激酶（AMPK）、過氧化物酶體增殖物激活受體α（PPARα）等一些與食欲調控和能量代謝相關的細胞因子反過來調控生物鐘。營養-生物鐘-能量代謝三者之間相互作用，使生物體適應環境的能力增強，能量利用達到最優。因此，通過調整進食時間和食物組分（如飼料能量水平和鈣水平），能夠改變能量代謝從而調節生物鐘的功能。將環境（光照管理）和營養（飼喂時間、飼料配方）綜合研究并加以運用，使機體生物鐘成為連接外部環境信號和內部能量代謝的紐帶，既能響應外界環境刺激，又能同時調控機體能量代謝進程，從而使各項生理功能得到更好地發揮，這將為蛋雞的產蛋調控機制研究提供新的視角。

生物鐘；蛋雞；產蛋；光照；能量

晝夜節律是生命的基本特征之一，它幾乎影響了生物體生命活動的方方面面，幫助其實現自身和外界環境的同步和適應。在自然狀態下, 生物鐘接受外界光/暗、食物、溫度以及化學因子等環境信號，調整自身節律保持與外界環境的同步[1-5]，從而適應環境。蛋雞的排卵-產蛋循環具有明顯的節律性和環境適應性。家禽在解剖學和生理學上與哺乳動物有很大的不同，性成熟的蛋雞卵巢內含有大量各種級別和各種狀態的卵泡。卵泡的成熟、排卵和蛋的形成是多組織、多過程、多層次參與的生理事件，在此過程中不同生理進程在時間上的吻合顯示了機體自身以及機體與環境之間的協調統一。已有的研究表明，內分泌激素的生成、禁食/采食、葡萄糖和脂質代謝、體溫的維持等一系列的行為和生理過程都有生物鐘參與調控[6-9]。生物鐘可使生物體預見環境的改變，從而調整它們的行為和生理機能來適應每天的環境變化。在哺乳動物中，中樞生物鐘位于下丘腦前段的視交叉上核（suprachiasmatic nucleus, SCN），外周生物鐘（peripheral clock）幾乎遍布全身各組織器官。中樞和外周生物鐘組成了一個有等級梯度的生物鐘系統，他們既相對獨立又互相聯系，共同維護機體各項生命活動的協調一致。哺乳動物的中樞生物鐘只有一個即SCN，而禽類的中樞生物鐘至少位于三處，分別是松果體、視網膜和SCN[10-11]。禽類和哺乳動物的生物節律分子機制高度保守[12]。筆者從晝夜生物鐘系統切入，整合生命過程中的節律現象，能夠全面地解析排卵-產蛋這一復雜又特殊的生理過程。因此，探索生物鐘系統在蛋雞生殖系統中的調控作用對于提高蛋雞產量、揭示生物體對外在環境的適應機制都有著重要意義。

1 生物鐘通過HPG軸影響排卵

動物的生殖系統發育和功能維持受到下丘腦-垂體-性腺（HPG）軸的調控。HPG軸啟動后，首先，下丘腦合成分泌促性腺激素釋放激素（GnRH）；其次，GnRH與受體結合，刺激垂體釋放促性腺激素，包括促黃體生成素（LH）和促卵泡激素（FSH）；最后，促性腺激素激活性腺的發育和性類固醇激素的分泌，如雌二醇和睪酮。下丘腦、垂體、性腺在中樞神經系統的調控下形成一個封閉的自動反饋系統，三者相互協調、相互制約使動物的生殖分泌系統保持相對穩定。

排卵最主要的誘發因素是來自垂體的LH峰。LH受體（LHR）的表達分布顯示，等級前卵泡和剛剛進入等級序列的F6和F5卵泡上LHR均較少，而在F1中最高。LH峰的釋放可以追溯到上游GnRH峰的生成，GnRH峰的定時性釋放在多種動物中都有過報道[13]。早期研究發現，切除SCN后性腺軸上的激素失去了正常狀態時的晝夜節奏性，并且擾亂了排卵的正常發生，這證明SCN參與了排卵調控。SCN處衍生出兩種神經元直接連接到GnRH神經元[14]，其中一種神經元-前腹側室周核神經元（AVPV），是雌激素反饋信號與晝夜節律信號的匯合點，即AVPV不僅受SCN晝夜節律性調控還可接受性激素的反饋性調控，使下游的GnRH峰表現為在特定時期（由生物鐘調控）由雌激素觸發（由性激素反饋性調控）的現象[15]。除SCN外，由禽類另一處中樞，即生物鐘-松果體合成分泌的褪黑素會直接作用于促性腺激素抑制激素（GnIH）神經元并調控GnIH的生成，GnIH既可以作用于GnRH神經元又可直接作用于腦垂體，從而抑制FSH和LH峰的生成。

中樞生物鐘可以直接感受外部環境信號的刺激[16]，并通過下游的神經內分泌系統向相應的靶組織傳遞輸出信號，所以說，外周生物鐘接受中樞的同步化信號來維持生物節律。卵巢中存在外周生物鐘己經在多個物種上被報道過[17]，但是卵巢是一個多組分的復雜組織，禽類尤其如此。研究發現，蛋禽排卵前卵泡F1-F3的顆粒細胞受到生物節律的直接調控，并在F1中節律震蕩最為強烈[18]。LH、FSH都可以影響小鼠卵巢顆粒細胞中生物鐘基因的表達[19-20]，而在禽類上的研究發現，只有LH具有同樣的作用[21]。

光照是影響動物繁殖的主要的環境調控因子[22]，光信號通過顱骨和視網膜，通過一系列神經信號轉導引起下丘腦血管活性腸肽（VIP）和催乳素（PRL）分泌上升，最終通過影響下丘腦GnRH和垂體FSH和LH來調控繁殖活動[23]。禽類的繁殖活動對光照是很敏感的，Hahn等[24]研究證明，將成年家雀由16L﹕8D的光照環境轉移到13D﹕11D后，下丘腦視前區和正中隆起的GnRH神經元與神經纖維增多，表明鳥類下丘腦GnRH的表達受光照時間的影響。長光照使鳥類腦內的GnRH表達以及外周血中LH和FSH的含量顯著下降[25]。光周期也能引起家禽PRL的分泌和濃度的改變[26]，隨著光照時間的增長，處于繁殖期的家禽PRL分泌不斷上升[27]。上述研究表明，光信號通過調控生物鐘影響HPG軸，從而調控機體的繁殖活動。

2 生物鐘整合能量/物質代謝影響產蛋

除了受到HPG的神經內分泌調控之外，蛋雞的排卵-產蛋過程還受到機體能量代謝的影響。卵泡吸收的卵黃來源于肝臟合成的卵黃前體物質-極低密度脂蛋白（VLDL）和卵黃蛋白原（VTG）。肝臟合成卵黃前體物質后, 經血液轉運至卵巢。卵泡中的初級卵母細胞不斷聚集卵黃，使卵泡體積增大，經成熟分化后排出。蛋形成時要分泌大量的卵清蛋白并動員大量的鈣去形成蛋殼，營養或鈣的缺乏可能會延長該過程。研究也發現，在能量缺乏時（禁食狀態或采食基礎日糧），應激激素抑制卵泡發育和產蛋性能；而在能量充足時（飼喂狀態或采食高脂日糧），這種作用會減弱[28]。這表明蛋雞的卵泡發育和產蛋性能與機體的能量狀態有關。最近的研究發現，產蛋鴨卵巢的生物鐘基因表達水平與產蛋量密切相關[29]，產蛋雞漏斗部（捕獲蛋黃的部位）和子宮部（形成蛋殼的部位）的生物鐘基因Bmal1、Clock、Per2和Per3在排卵過程中發揮了重要作用[30]。

2.1 生物鐘調控能量攝入

隨著一天中能量需求的波動，動物的采食行為也呈現節律性。研究發現，幾乎80%的食物消耗于小鼠活躍的夜間。禽類胃腸道長度相對較短，食糜通過消化道速度較快，因此禽類有頻繁采食的習性，其累積采食量較高。自然光照下，雞采食高峰發生在清晨和黃昏[31]。通過調整光照改變晝夜節律，能夠調節雞的采食量[32]，這表明生物體的鐘基因調控著食欲和采食行為。研究證明，中樞和外周的生物鐘基因Bmal1能夠調控食欲調節系統[33-34]。穹隆周區和下丘腦背內側核的食欲素（orexin）神經元具有晝夜節律性活動[35-36]，穹隆周區orexin神經元受視交叉上核谷氨酸能和γ-氨基丁酸能神經元的支配，而視交叉上核同時也是生物鐘的中樞調節部位；進一步研究證明，orexin神經元還參與了睡眠/覺醒周期、食欲、自主神經活動以及晝夜節律性的調節[37-38]。雞與哺乳動物的orexin同源性很高[39]。在哺乳動物中，瘦素在機體食欲調控和能量代謝中發揮著重要作用，敲除SCN會破壞瘦素表達的節律性[40]。小鼠敲除生物鐘基因Bmal1后導致瘦素的分泌和基因表達發生改變[41]。敲除生物鐘基因Clock后，與食欲調控有關的神經肽orexin和胃饑餓素ghrelin 的 mRNA 表達水平均下降[42-43]。

代謝物和進食行為也可以反過來調控生物鐘[44]，其中進食時間可能比食物組分更重要[45-46]。在小鼠不活躍的光照時期給予它們食物，此時能量消耗低、呼吸交換率高，將導致生物時鐘的不同步以及代謝紊亂[47-48]。攝食時間的改變使外周生物鐘基因與中樞生物鐘基因表達的相位發生解偶聯[45]。對喪失了基本生物節律的小鼠在特定的時間給予食物，可以恢復其肝臟中某些基因表達的節律性[49]。雞上的研究也發現，限飼能夠改變雞的生物節律和活動[50]。采食時間和采食行為對生物鐘的影響可能是通過一些與食欲調控和能量代謝相關的細胞因子實現的。研究發現，食物消耗可能通過腺苷酸活化蛋白激酶（AMPK）改變生物鐘基因的表達，AMPK作為細胞能量感受器，在缺失時將導致肝臟中生物鐘基因Cry1的穩定性和時鐘節律性消失[51]。此外，食物還可能通過瘦素影響生物鐘。小鼠肝臟和脂肪組織中缺失瘦素，其正常活動和時鐘基因表達節律減弱[52]，瘦素受體缺失的小鼠在脂肪組織中也表現出生物鐘基因功能損傷[53]，補充瘦素能夠恢復生物鐘的功能并改善代謝指標[52]。

2.2 生物鐘調控能量代謝

生物鐘可以調控機體多種代謝途徑，它能有效調節整個代謝過程及相關信號以及組織的代謝功能。研究發現，能量代謝活躍的外周組織如肝臟、骨骼肌、脂肪組織中約有5%—10%的基因都呈節律表達，并且具有明顯的組織特異性[47, 54]。與能量代謝相關的激素，如胰島素、脂聯素、腎上腺糖皮質激素、瘦素等[55]，能量代謝相關酶的表達和活性[56]，以及與糖脂代謝相關的核受體大多也呈節律表達[57]。禽類的血漿葡萄糖、甘油三酯和肌酐也呈現明顯的晝夜節律性[58]。生物鐘基因在上述代謝過程中發揮著重要的調控作用，其中在與卵泡發育密切相關的脂質穩態調控中，Clock 和Bmal1扮演著重要角色。Zvonic等[47]研究表明，20%以上的小鼠脂肪轉錄組表達受晝夜節律調控。機體通過調節Clock 和 Bmal1，能夠驅動脂肪代謝關鍵酶ATGL和HSL的節律性表達[59]，使循環中游離脂肪酸水平保持節律性[59-61]；Bmal1的mRNA 水平在脂肪分化的過程中高度表達[62]，Bmal1通過激活視黃酸相關孤兒核受體α（RORα）調節骨骼肌的脂肪生成和貯存[63]，在Bmal1全身性敲除的小鼠中，瘦素、脂聯素、抵抗素等脂肪細胞因子的分泌和基因的表達均發生改變[41]；Clock突變的小鼠比正常小鼠更胖, 并伴有高血脂、脂肪肝等癥狀，這主要歸因于脂肪的沉積和脂肪細胞肥大[60]。雞上的研究也發現，生物鐘影響脂肪合成[64]，與脂肪合成密切相關的轉錄因子膽固醇調節元件結合蛋白（SREBP）及其下游的靶基因，也受到光照和生物鐘的調控[65]。

生物鐘對代謝過程的調控通過以下方式實現：①調節代謝途徑中重要限速酶的表達。如膽固醇生物合成的限速酶HMG-CoA還原酶（HMGCR）的激活呈現節律性[66-67]。②整合核受體和營養信號蛋白。如過氧化物酶體增殖物激活受體α（PPARα）是脂肪代謝主要調節因子，生物鐘基因Clock 和 Bmal1能夠結合到PPARα啟動子的 E-box上，直接調節PPARα的表達[68]；生物鐘基因Per2與核受體REV-ERBα相互作用從而調控肝臟糖代謝[69]；生物鐘基因Per3通過結合到PPARγ的靶位點來抑制其表達，從而阻礙脂肪生成[70]。③調節代謝感受器和代謝物。Minami等[71]研究表明，數百種代謝物的含量水平在小鼠胞質中表現出晝夜振蕩，包括磷脂、氨基酸和尿素循環的中間產物；AMPK是細胞能量狀態的感受器，在小鼠的肝臟、下丘腦等組織中，AMPK的活性也是有節律的[72]。

另一方面，能量代謝也可以反過來調控生物鐘。如過氧化物酶體增殖物激活受體γ共激活因子α（PGC-1α）受控于生物鐘，反過來它又可調控生物鐘，是連接生物鐘和能量代謝的重要調控因子[73]。核受體PPARα能夠結合到Bmal1啟動子的PPARα反應元件（PPRE）上，調控Bmal1的表達[68]。能量感受器AMPK可以通過磷酸化 Cry1[51]和 Ck1ε[74]來調節生物時鐘。一些原本是生物鐘的輸出信號，也可作為后續時鐘循環的輸入信號，如cAMP和NAD+[75-77]。

此外，體內的能量狀態也可以通過代謝信號反過來作用于生物鐘。研究發現，營養水平直接影響SCN的時相。給予高脂飼料的小鼠，其生物節律發生改變，自發活動周期延長[78-79]，高膽固醇飲食不影響肝臟中生物鐘基因（和）以及鐘控基因（和）的節律表達, 但會使生物鐘控制基因Pai-1的表達量上升[80]。高脂飲食能夠顯著抑制小鼠脂肪組織中時鐘關鍵基因、及的表達[79]。飼喂低能飼料的雞，其生物節律發生改變，活動減少[81]。PGC-1α和PPARα是連接生物鐘和能量代謝的重要調控因子[68, 73]，因此推測，代謝物和進食行為對生物鐘基因的影響可能是通過PGC- 1α和PPARα實現的。

近年來的研究發現，在生物鐘和能量代謝的互作網絡中，腸道微生物扮演了重要角色[82]。由大量微生物菌群組成的腸道微環境參與了機體的免疫調控及能量代謝等生理過程[83]。腸道內的微生物與宿主相互作用，共同維持機體動態的生物平衡。研究發現，腸道微生物也會受到生物鐘的調控，這些腸道微生物的生物節律與其宿主具有同步性[84-89]。研究發現，在大鼠活躍的暗周期，腸道微生物主要負責消化營養物質、修復并延伸其DNA；在大鼠不活躍的亮周期，腸道微生物主要參與排毒、感知環境信號、長出鞭毛輔助移動等進程[90]。進一步研究發現，腸道微生物的這種節律性與生物鐘基因Per1/2的調控有關[90]。腸道微生物的區系和多樣性均具有生物節律[91]，并且會影響到機體代謝物、肝臟轉錄組和解毒功能的生物節律[92]，影響肝臟功能的節律性[93]。高脂飲食能干擾腸道微生物的這種節律，反過來，對腸道微生物的節律進行調控能改善因高脂飲食導致的肥胖[94]。因此，腸道微生物能同時響應并調控生物鐘和能量代謝過程。

2.3 生物鐘調控鈣代謝

蛋殼的主要成分是碳酸鈣，蛋雞可從骨組織中動員8%—10%的鈣用于形成蛋殼，所以鈣在骨組織中的動員和在蛋殼腺中的沉積對蛋的形成非常重要。雞蛋蛋殼的形成具有明顯的生物節律，蛋殼形成的最活躍時期常處于光照周期的黑暗階段。骨代謝的平衡也與生物鐘基因的調控和支配有關，成骨細胞具有生物鐘基因，其增殖活性表現為明顯的晝低夜高的24h節律變化，這表明機體鈣代謝是受到生物鐘調控的。

松果體作為禽類的中樞生物鐘之一，其分泌的褪黑素在主導生物節律、調控骨的代謝平衡和鈣代謝方面具有重要作用。褪黑素可以直接作用于破骨細胞、成骨細胞及直接調節鈣代謝平衡，或者通過增加非快動眼睡眠時相，增加生長激素的分泌，從而間接影響骨代謝。研究表明褪黑素可通過介導降鈣素、甲狀旁腺素（PTH）及雌激素分泌來調節體內鈣代謝[95]。Conti等[96]研究發現骨髓細胞中含有高濃度的褪黑素，并對骨髓細胞增殖有積極作用。骨髓的褪黑素水平為夜間血漿褪黑素水平的2 倍[97]。蛋雞上的研究也發現，褪黑素能調節鈣的分配，從而影響骨強度和蛋殼重量[98]。除褪黑素外，PTH也是與鈣代謝相關激素中研究最多的激素之一。研究顯示，在生理情況下，PTH的分泌具有晝夜節律性，高峰出現在上午的0—6點[99]。PTH對鈣代謝的影響主要表現為節律的紊亂，用磷酸鹽或鈣制劑進行時間療法可以調整內源性PTH激素的晝夜節律，鈣代謝紊亂也隨之顯著改善[100]。在產蛋期尤其是產蛋后期，產蛋雞對鈣的需要量增加，夜間補充光照和補充飼喂次數有利于雞群在形成蛋殼期間攝取飼料中的鈣，提高產蛋率、改善蛋殼質量[101]。

綜上所述，營養-生物鐘-能量代謝，三者之間相互作用，使生物體適應環境的能力增強，能量利用達到最優。因此，可以通過調整進食時間和食物組分（如飼料能量水平和鈣水平），改變能量代謝從而調節生物鐘的功能。

3 結論與展望

目前我國蛋雞養殖逐漸趨向規模化、集約化，蛋品市場也向品牌化方向發展。市場對雞蛋質量的要求越來越高，尤其是在產蛋后期，蛋雞機體老化和飼養管理落后等因素都會造成雞蛋品質的下降。光照是影響家禽繁殖和生產的最重要的生態因子之一，光信號作用于中樞生物鐘，通過神經內分泌機制影響HPG軸來調控繁殖活動。另外，食物組分以及進食時間可以顯著調控機體的生物鐘。因此，了解飼糧中各種營養素及進食時間與生物鐘的相互關系，將環境（光照管理）和營養（飼喂時間、飼料配方）綜合研究并加以運用，使機體生物鐘成為連接外部信號和內部能量代謝的紐帶，既能響應上游環境刺激，又能同時調控下游能量代謝進程，從而使各項生理功能得到更好地發揮，這將為蛋雞的產蛋調控機制研究提供新的視角。

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（責任編輯 林鑒非）

Regulation of Biological Clock in Ovulation-Laying of Laying Hens

WANG XiaoJuan, LIU Lei, JIAO HongChao, ZHAO JingPeng, LIN Hai

(Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Department of Animal Science, Shandong Agricultural University, Tai’an 271018)

The endogenous circadian rhythm enables the organisms to predict the changes of environmental cycle, which maintains consistency between body metabolism and the external environment. During the maturation of follicular, ovulation, and the formation of egg in birds, the coincidence of the different physiological processes in time shows the unity of the body itself and the coordination between the body and the environment. Biological clock participates in a series of behavior and physiological processes such as nutrition intake, the production of endocrine hormones and energy metabolism. In the present review, the role of biological clock in neuroendocrine, energy intake and energy metabolism has been discussed, from the points of light factor and nutrition factor, to reveal the potential regulating mechanism underlying ovulation and egg laying of hens. (1) Light signal acts on hypothalamic- pituitary-gonadal axis (HPG) by regulating the biological clock to influence reproductive activities. Under the stimulation of light, the central clocks in suprachiasmatic nucleus (SCN) and pineal act on hypothalamus, and make it to release gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormones (GnIH) periodically. GnRH and GnIH then act on pituitary, and make it to release gonadotropin hormone, that is luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Periphery clocks in ovary receive the central synchronization signal to maintain the biological rhythm, thereby regulating the maturation of follicles and ovulation. (2) In addition to being regulated by the neuroendocrine system of HPG axis, the ovulation-egg production process of laying hens is also affected by the body's energy metabolism. The central and peripheral clock genes regulate the appetite regulation system and thus affect energy intake; Biological clock can regulate the expression of key enzymes in the process of metabolism, integrate the nuclear receptors and nutrition signaling proteins, regulate metabolism sensors and metabolites, affect gut microbes to regulate energy metabolism, and affect the synthesis, transport and deposition of yolk precursor;Melatonin secreted by bird's pineal can regulate calcium metabolism rhythmically by mediating the secretion of calcitonin, parathyroid hormone (PTH) and estrogen, and influence the formation of egg shell.The time and the behavior of energy intake, the body energy metabolism and energy status can also modulate biological clock, through some appetite regulation and energy metabolism related cytokines such as AMP-activated protein kinase (AMPK), and peroxisome proliferator-activated receptors α (PPARα). There are interactions between nutrient, biological clock and energy metabolism, which accommodate organisms with the surrounding and optimize the energy utilization. Therefore, by adjusting the time of eating and the composition of feed (such as the energy level of feed and calcium level), energy metabolism can be changed to regulate the function of the biological clock. In conclusion, it will provide a new perspective for researching regulation mechanism of egg laying, if we make an integrated study on environment factor (light management) and nutrition (feeding time and feed formula) in which biological clock linked external factors and internal energy metabolism, that is, biological clock can both response to environmental stimuli, and regulate the body's energy metabolism process, to optimize the various physiological functions.

biological clock; laying hen; egg laying; light; energy

2018-04-04；

2018-06-13

“十三五”國家重點研發計劃（2016YFD0500510）、國家自然科學基金（31672441）、國家現代農業產業技術體系建設專項資金（CARS-41）、山東省“雙一流”獎補資金、泰山學者項目（201511023）

王曉鵑，E-mail：wangxj@sdau.edu.cn。

林海，E-mail：hailin@sdau.edu.cn